Nucleotide hemi-sulfate salt for the treatment of hepatitis c virus

ABSTRACT

to treat a host infected with hepatitis C, as well as pharmaceutical compositions and dosage forms, including solid dosage forms, thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/885,630 filed Jan. 31, 2018, which claims the benefit of provisionalU.S. Application No. 62/453,437 filed Feb. 1, 2017; 62/469,912 filedMar. 10, 2017; 62/488,366 filed Apr. 21, 2017; and, 62/575,248 filedOct. 20, 2017. The entirety of these applications are incorporated byreference herein for all purposes.

FIELD OF THE INVENTION

The present invention is the hemi-sulfate salt of a selected nucleotidecompound that has unexpected therapeutic properties to treat a hostinfected with hepatitis C, as well as pharmaceutical compositions anddosage forms thereof.

BACKGROUND OF THE INVENTION

Hepatitis C (HCV) is an RNA single-stranded virus and member of theHepacivirus genus. It is estimated that 75% of all cases of liverdisease are caused by HCV. HCV infection can lead to cirrhosis and livercancer, and if left to progress, liver failure that may require a livertransplant. Approximately 71 million people worldwide are living withchronic HCV infections and approximately 399,000 people die each yearfrom HCV, mostly from cirrhosis and hepatocellular carcinoma.

RNA polymerase is a key target for drug development against RNA singlestranded viruses. The HCV non-structural protein NS5B RNA-dependent RNApolymerase is a key enzyme responsible for initiating and catalyzingviral RNA synthesis. There are two major subclasses of NS5B inhibitors:nucleoside analogs and non-nucleoside inhibitors (NNIs). Nucleosideanalogs are anabolized to active triphosphates that act as alternativesubstrates for the polymerase and non-nucleoside inhibitors (NNIs) bindto allosteric regions on the protein. Nucleoside or nucleotideinhibitors mimic natural polymerase substrates and act as chainterminators. They inhibit the initiation of RNA transcription andelongation of a nascent RNA chain.

In addition to targeting RNA polymerase, other RNA viral proteins mayalso be targeted in combination therapies. For example, HCV proteinsthat are additional targets for therapeutic approaches are NS3/4A (aserine protease) and NS5A (a non-structural protein that is an essentialcomponent of HCV replicase and exerts a range of effects on cellularpathways).

In December 2013, the first nucleoside NS5B polymerase inhibitorsofosbuvir (Sovaldi®, Gilead Sciences) was approved. Sovaldi® is auridine phosphoramidate prodrug that is taken up by hepatocytes andundergoes intracellular activation to afford the active metabolite,2′-deoxy-2′-α-fluoro-β-C-methyluridine-5′-triphosphate.

Sovaldi® is the first drug that has demonstrated safety and efficacy totreat certain types of HCV infection without the need forco-administration of interferon. Sovaldi® is the third drug withbreakthrough therapy designation to receive FDA approval.

In 2014, the U.S. FDA approved Harvoni® (ledispasvir, a NS5A inhibitor,and sofosbuvir) to treat chronic hepatitis C virus Genotype 1 infection.Harvoni® is the first combination pill approved to treat chronic HCVGenotype 1 infection. It is also the first approved regimen that doesnot require administration with interferon or ribavirin. In addition,the FDA approved simeprevir (Olysio™) in combination with sofosbuvir(Sovaldi®) as a once-daily, all oral, interferon and ribavirin-freetreatment for adults with Genotype 1 HCV infection.

The U.S. FDA also approved AbbVie's VIEKIRA Pak™ in 2014, a multi-pillpack containing dasabuvir (a non-nucleoside NS5B polymerase inhibitor),ombitasvir (a NS5A inhibitor), paritaprevir (a NS3/4A inhibitor), andritonavir. The VIEKIRA Pak™ can be used with or without the ribavirin totreat Genotype 1 HCV infected patients including patients withcompensated cirrhosis. VIEKIRA Pak™ does not require interferonco-therapy.

In July 2015, the U.S. FDA approved Technivie™ and Daklinza™ for thetreatment of HCV genotype 4 and HCV Genotype 3, respectively. Technivie™(Ombitasvir/paritaprevir/ritonavir) was approved for use in combinationwith ribavirin for the treatment of HCV genotype 4 in patients withoutscarring and cirrhosis and is the first option for HCV-4 infectedpatients who do not require co-administration with interferon. Daklinza™was approved for use with Sovaldi® to treat HCV genotype 3 infections.Daklinza™ is the first drug that has demonstrated safety and efficacy intreating HCV Genotype 3 without the need for co-administration ofinterferon or ribavirin.

In October 2015, the U.S. FDA warned that HCV treatments Viekira Pak andTechnivie can cause serious liver injury primarily in patients withunderlying advanced liver disease and required that additionalinformation about safety be added to the label.

Other current approved therapies for HCV include interferon alpha-2b orpegylated interferon alpha-2b (Pegintron®), which can be administeredwith ribavirin (Rebetor®), NS3/4A telaprevir (Incivek®, Vertex andJohnson & Johnson), boceprevir (Victrelis™, Merck), simeprevir (Olysio™,Johnson & Johnson), paritaprevir (AbbVie), Ombitasvir (AbbVie), the NNIDasabuvir (ABT-333) and Merck's Zepatier™ (a single-tablet combinationof the two drugs grazoprevir and elbasvir).

Additional NS5B polymerase inhibitors are currently under development.Merck is developing the uridine nucleotide prodrug MK-3682 (formerlyIdenix IDX21437) and the drug is currently in Phase II combinationtrials.

United States patents and WO applications that describe nucleosidepolymerase inhibitors for the treatment of Flaviviridae, including HCV,include those filed by Idenix Pharmaceuticals (U.S. Pat. Nos. 6,812,219;6,914,054; 7,105,493; 7,138,376; 7,148,206; 7,157,441; 7,163,929;7,169,766; 7,192,936; 7,365,057; 7,384,924; 7,456,155; 7,547,704;7,582,618; 7,608,597; 7,608,600; 7,625,875; 7,635,689; 7,662,798;7,824,851; 7,902,202; 7,932,240; 7,951,789; 8,193,372; 8,299,038;8,343,937; 8,362,068; 8,507,460; 8,637,475; 8,674,085; 8,680,071;8,691,788, 8,742,101, 8,951,985; 9,109,001; 9,243,025; U.S.2016/0002281; U.S. 2013/0064794; WO/2015/095305; WO/2015/081133;WO/2015/061683; WO/2013/177219; WO/2013/039920; WO/2014/137930;WO/2014/052638; WO/2012/154321); Merck (U.S. Pat. Nos. 6,777,395;7,105,499; 7,125,855; 7,202,224; 7,323,449; 7,339,054; 7,534,767;7,632,821; 7,879,815; 8,071,568; 8,148,349; 8,470,834; 8,481,712;8,541,434; 8,697,694; 8,715,638, 9,061,041; 9,156,872 andWO/2013/009737); Emory University (U.S. Pat. Nos. 6,348,587; 6,911,424;7,307,065; 7,495,006; 7,662,938; 7,772,208; 8,114,994; 8,168,583;8,609,627; U.S. 2014/0212382; and WO2014/1244430); GileadSciences/Pharmasset Inc. (U.S. Pat. Nos. 7,842,672; 7,973,013;8,008,264; 8,012,941; 8,012,942; 8,318,682; 8,324,179; 8,415,308;8,455,451; 8,563,530; 8,841,275; 8,853,171; 8,871,785; 8,877,733;8,889,159; 8,906,880; 8,912,321; 8,957,045; 8,957,046; 9,045,520;9,085,573; 9,090,642; and 9,139,604) and (6,908,924; 6,949,522;7,094,770; 7,211,570; 7,429,572; 7,601,820; 7,638,502; 7,718,790;7,772,208; RE42,015; 7,919,247; 7,964,580; 8,093,380; 8,114,997;8,173,621; 8,334,270; 8,415,322; 8,481,713; 8,492,539; 8,551,973;8,580,765; 8,618,076; 8,629,263; 8,633,309; 8,642,756; 8,716,262;8,716,263; 8,735,345; 8,735,372; 8,735,569; 8,759,510 and 8,765,710);Hoffman La-Roche (U.S. Pat. No. 6,660,721), Roche (U.S. Pat. Nos.6,784,166; 7,608,599, 7,608,601 and 8,071,567); Alios BioPharma Inc.(U.S. Pat. Nos. 8,895,723; 8,877,731; 8,871,737, 8,846,896, 8,772,474;8,980,865; 9,012,427; U.S. 2015/0105341; U.S. 2015/0011497; U.S.2010/0249068; U.S. 2012/0070411; WO 2015/054465; WO 2014/209979; WO2014/100505; WO 2014/100498; WO 2013/142159; WO 2013/142157; WO2013/096680; WO 2013/088155; WO 2010/108135), Enanta Pharmaceuticals (US8,575,119; 8,846,638; 9,085,599; WO 2013/044030; WO 2012/125900), Biota(U.S. Pat. Nos. 7,268,119; 7,285,658; 7,713,941; 8,119,607; 8,415,309;8,501,699 and 8,802,840), Biocryst Pharmaceuticals (U.S. Pat. Nos.7,388,002; 7,429,571; 7,514,410; 7,560,434; 7,994,139; 8,133,870;8,163,703; 8,242,085 and 8,440,813), Alla Chem, LLC (U.S. Pat. No.8,889,701 and WO 2015/053662), Inhibitex (U.S. Pat. No. 8,759,318 andWO/2012/092484), Janssen Products (U.S. Pat. Nos. 8,399,429; 8,431,588,8,481,510, 8,552,021, 8,933,052; 9,006,29 and 9,012,428) the Universityof Georgia Foundation (U.S. Pat. Nos. 6,348,587; 7,307,065; 7,662,938;8,168,583; 8,673,926, 8,816,074; 8,921,384 and 8,946,244), RFS Pharma,LLC (U.S. Pat. Nos. 8,895,531; 8,859,595; 8,815,829; 8,609,627;7,560,550; U.S. 2014/0066395; U.S. 2014/0235566; U.S. 2010/0279969;WO/2010/091386 and WO 2012/158811) University College CardiffConsultants Limited (WO/2014/076490, WO 2010/081082; WO/2008/062206),Achillion Pharmaceuticals, Inc. (WO/2014/169278 and WO 2014/169280),Cocrystal Pharma, Inc. (U.S. Pat. No. 9,173,893), KatholiekeUniversiteit Leuven (WO 2015/158913), Catabasis (WO 2013/090420) and theRegents of the University of Minnesota (WO 2006/004637).

Atea Pharmaceuticals, Inc. has disclosedβ-D-2′-deoxy-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-(mono- anddi-methyl) purine nucleotides for the treatment of HCV in U.S. Pat. No.9,828,410 and PCT Application No. WO 2016/144918. Atea has alsodisclosedβ-D-2′-deoxy-2′-substituted-4′-substituted-2-N⁶-substituted-6-aminopurinenucleotides for the treatment of paramyxovirus and orthomyxovirusinfections in U.S. 2018/0009836 and WO 2018/009623.

There remains a strong medical need to develop anti-HCV therapies thatare safe, effective and well-tolerated. The need is accentuated by theexpectation of drug resistance. More potent direct-acting antiviralscould significantly shorten treatment duration and improve complianceand SVR (sustained viral response) rates for patients infected with allHCV genotypes.

It is therefore an object of the present invention to provide compounds,pharmaceutical compositions, methods, and dosage forms to treat and/orprevent infections of HCV.

SUMMARY OF THE INVENTION

It has been surprisingly discovered that the hemisulfate salt ofCompound 1, which is provided below as Compound 2, exhibits unexpectedadvantageous therapeutic properties, including enhanced bioavailabilityand target organ selectivity, over its free base (Compound 1). Theseunexpected advantages could not have been predicted in advance. Compound2 is thus a therapeutically superior composition of matter to administerin an effective amount to a host in need thereof, typically a human, forthe treatment of hepatitis C. Compound 2 is referred to as thehemi-sulfate salt ofisopropyl((S)-(((2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate.Compound 1 is disclosed in U.S. Pat. No. 9,828,410.

Compound 2, as Compound 1, is converted to its correspondingtriphosphate nucleotide (Compound 1-6) in the cell, which is the activemetabolite and inhibitor of RNA polymerase (see Scheme 1 below). SinceCompound 1-6 is produced in the cell and does not leave the cell, it isnot measurable in the plasma. However, the 5′-OH metabolite Compound 1-7(see Scheme 1) is exported from the cell, and therefore is measurable inplasma and acts as a surrogate of the concentration of intracellularactive metabolite Compound 1-6.

It has been discovered that the plasma concentration in vivo ofsurrogate Compound 1-7, and thus intracellular Compound 1-6, issubstantially higher when Compound 2 is administered in vivo than whenCompound 1 is administered in vivo. In a head-to-head comparison of dogsdosed with Compound 1 and Compound 2 (Example 19, Table 28), dosing withCompound 2 achieved an AUC_((0-4hrs)) of the ultimate guanine 5′-OHnucleoside metabolite (1-7) that is twice as high as the AUC followingCompound 1 dosing. It is unexpected that a non-covalent salt has such aneffect on plasma concentration of the parent drug (Compound 1).

Additionally, Compound 2 selectively partitions in vivo to the liverover the heart (Example 19, Table 29), which is beneficial since theliver is the diseased organ in hosts infected with HCV. Dogs were dosedwith Compound 1 or Compound 2 and the concentration of the activetriphosphate (1-6) in the liver and heart was measured. The liver toheart ratio of the active triphosphate concentration was higher afterdosing with Compound 2 compared to Compound 1 as shown in Table 29.Specifically, the liver/heart partitioning ratio for Compound 2 is 20compared to a liver/heart partitioning ratio of 3.1 for Compound 1. Thisdata indicates, unexpectedly, that the administration of Compound 2results in the preferential distribution of the active guaninetriphosphate (Compound 1-6) in the liver over the heart when compared toCompound 1, which reduces potential off-target effects. It wasunexpected that administration of Compound 2 would significantly reduceundesired off-target partitioning. This allows for the administration ofCompound 2 at a higher dose than Compound 1, if desired by thehealthcare practitioner.

In addition, liver and heart tissue levels of the active guaninetriphosphate derivative of Compound 2 (metabolite 1-6) were measuredafter oral doses of Compound 2 in rats and monkeys (Example 20). Highlevels of the active guanine triphosphate (1-6) were measured in theliver of all species tested. Importantly, unquantifiable levels of theguanine triphosphate (1-6) were measured in monkey hearts, and this isindicative of liver-specific formation of the active triphosphate. Itwas thus discovered that compared to Compound 1 dosing, Compound 2dosing improves guanine triphosphate (1-6) distribution.

When administered to healthy and hepatitis C infected patients, Compound2 was well tolerated after a single oral dose and C_(max), T_(max) andAUC_(tot) pharmacokinetic parameters were comparable in both groups(Tables 34 and 35). As described in Example 24, a single dose ofCompound 2 in HCV-infected patients resulted in a significant antiviralactivity. Plasma exposure of metabolite 1-7 was mostly dose-proportionalover the studied range.

Individual pharmacokinetic/pharmacodynamic analyses of patients dosedwith Compound 2 showed that the viral response correlated with plasmaexposure of metabolite 1-7 of Compound 2 (Example 24, FIGS. 23A-23F),indicating that profound vial responses are achievable with robust dosesof Compound 2.

Example 24 confirms that, as non-limiting embodiments, single oral dosesof 300 mg, 400 mg, and 600 mg result in significant antiviral activityin humans. The C₂₄ trough plasma concentration of metabolite 1-7following a 600 mg dose of Compound 2 doubled from the C₂₄ trough plasmaconcentration of metabolite 1-7 following a 300 mg dose of Compound 2.

FIG. 24 and Example 25 highlight the striking invention provided byCompound 2 for the treatment of hepatitis C. As shown in FIG. 24, thesteady-state trough plasma levels (C_(24,ss)) of metabolite 1-7following Compound 2 dosing in humans (600 mg QD (550 mg free baseequivalent) and 450 mg QD (400 mg free base equivalent)) was predictedand compared to the EC₉₅ of Compound 1 in vitro across a range of HCVclinical isolates to determine if the steady state plasma concentrationis consistently higher than the EC₉₅, which would result in highefficacy against multiple clinical isolates in vivo. The EC₉₅ forCompound 1 is the same as the EC₉₅ of Compound 2. For Compound 2 to beeffective, the steady-state trough plasma level of metabolite 1-7 shouldexceed the EC₉₅.

As shown in FIG. 24, the EC₉₅ of Compound 2 against all tested clinicalisolates ranged from approximately 18 nM to 24 nM.

As shown in FIG. 24, Compound 2 at a dose of 450 mg QD (400 mg free baseequivalent) in humans provides a predicted steady state trough plasmaconcentration (C_(24,ss)) of approximately 40 ng/mL. Compound 2 at adose of 600 mg QD (550 mg free base equivalent) in humans provides apredicted steady state trough plasma concentration (C_(24,ss)) ofapproximately 50 ng/mL.

Therefore, the predicted steady state plasma concentration of surrogatemetabolite 1-7 is almost double the EC₉₅ against all tested clinicalisolates (even the hard to treat GT3a), which indicates superiorperformance.

In contrast, the EC₉₅ of the standard of care nucleotide sofosbuvir(Sovaldi) ranges from 50 nM to 265 nM across all tested HCV clinicalisolates, with an EC₉₅ less than the predicted steady stateconcentration at the commercial dosage of 400 mg for only two isolates,GT2a and GT2b. The EC₉₅ for the commercial dosage of 400 mg ofsofosbuvir is greater than the predicted steady state concentration forother clinical isolates, GT1a, GT1b, GT3a, GT4a, and GT4d.

The data comparing the efficacy and pharmacokinetic steady stateparameters in FIG. 24 clearly demonstrates the unexpected therapeuticimportance of Compound 2 for the treatment of hepatitis C. In fact, thepredicted steady-state (C_(24,ss)) plasma level after administration ofCompound 2 is predicted to be at least 2-fold higher than the EC₉₅ forall genotypes tested, and is 3- to 5-fold more potent against GT2. Thisdata indicates that Compound 2 has potent pan-genotypic antiviralactivity in humans. As shown in FIG. 24, the EC₉₅ of sofosbuvir againstGT1, GT3, and GT4 is greater than 100 ng/mL. Thus surprisingly, Compound2 is active against HCV at a dosage form that delivers a lowersteady-state trough concentration (40-50 ng/mL) than the steady-statetough concentration (approximately 100 ng/mL) achieved by the equivalentdosage form of sofosbuvir.

In one embodiment, therefore, the invention includes a dosage form ofCompound 2 that provides a metabolite 1-7 steady-state plasma troughconcentration (C_(24,ss)) between approximately 15-75 ng/mL, forexample, 20-60 ng/mL, 25-50 ng/mL, 40-60 ng/mL, or even 40-50 ng/mL.This is unexpected in light of the fact that the steady stateconcentration of the equivalent metabolite of sofosbuvir isapproximately 100 ng/mL.

Additionally, it has been discovered that Compound 2 is an unusuallystable, highly soluble, non-hygroscopic salt with activity against HCV.This is surprising because a number of salts of Compound 1 other thanthe hemi-sulfate salt (Compound 2), including the mono-sulfate salt(Compound 3), are not physically stable, but instead deliquesce orbecome gummy solids (Example 4), and thus are not suitable for stablesolid pharmaceutical dosage forms. Surprisingly, while Compound 2 doesnot become gummy, it is up to 43 times more soluble in water compared toCompound 1 and is over 6 times more soluble than Compound 1 undersimulated gastric fluid (SGF) conditions (Example 15).

As discussed in Example 16, Compound 2 remains a white solid with an IRthat corresponds to the reference standard for 6 months underaccelerated stability conditions (40° C./75% RH). Compound 2 is stablefor 9 months at ambient conditions (25° C./60% RH) and refrigeratorconditions (5° C.).

Solid dosage forms (50 mg and 100 mg tablets) of Compound 2 are alsochemically stable under accelerated (40° C./75% RH) and refrigerationconditions (5° C.) for 6 months (Example 26). Compound 2 is stable underambient conditions (25° C./60% RH) in a solid dosage form for at least 9months.

Scheme 1 provides the metabolic pathway of Compound 1 and Compound 2,which involves the initial de-esterification of the phosphoramidate(metabolite 1-1) to form metabolite 1-2. Metabolite 1-2 is thenconverted to the N⁶-methyl-2,6-diaminopurine-5′-monophosphate derivative(metabolite 1-3), which is in turn metabolized to the free5′-hydroxyl-N⁶-methyl-2,6-diaminopurine nucleoside (metabolite 1-8) and((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methyldihydrogen phosphate as the 5′-monophosphate (metabolite 1-4).Metabolite 1-4 is anabolized to the corresponding diphosphate(metabolite 1-5) and then the active triphosphate derivative (metabolite1-6). The 5′-triphosphate can be further metabolized to generate2-amino-9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one(1-7). Metabolite 1-7 is measurable in plasma and is therefore asurrogate for the active triphosphate (1-6), which is not measurable inplasma.

In one embodiment, the invention is Compound 2 and its use to treathepatitis C (HCV) in a host in need thereof, optionally in apharmaceutically acceptable carrier. In one aspect, Compound 2 is usedas an amorphous solid. In another aspect, Compound 2 is used as acrystalline solid.

The present invention further includes an exemplary on-limiting processfor the preparation of Compound 2 that includes

(i) a first step of dissolving Compound 1 in an organic solvent, forexample, acetone, ethyl acetate, methanol, acetonitrile, or ether, orthe like, in a flask or container;

(ii) charging a second flask or container with a second organic solvent,which may be the same as or different from the organic solvent in step(i), optionally cooling the second solvent to 0-10 degrees C., andadding dropwise H₂SO₄ to the second organic solvent to create aH₂SO₄/organic solvent mixture; and wherein the solvent for example maybe methanol;

(iii) adding dropwise the H₂SO₄/solvent mixture at a molar ratio of0.5/1.0 from step (ii) to the solution of Compound 1 of step (i) atambient or slightly increased or decreased temperature (for example23-35 degrees C.);

(iv) stirring the reaction of step (iii) until precipitate of Compound 2is formed, for example at ambient or slightly increased or decreasedtemperature;

(v) optionally filtering the resulting precipitate from step (iv) andwashing with an organic solvent; and

(vi) optionally drying the resulting Compound 2 in a vacuum, optionallyat elevated a temperature, for example, 55, 56, 57, 58, 59, or 60° C.

In one embodiment, the organic solvent in step (i) is3-methyl-2-pentanone. In one embodiment, the organic solvent in step (i)is ethyl isopropyl ketone. In one embodiment, the organic solvent instep (i) is methyl propionate. In one embodiment, the organic solvent instep (i) is ethyl butyrate.

Despite the volume of antiviral nucleoside literature and patentfilings, Compound 2 has not been specifically disclosed. Accordingly,the present invention includes Compound 2, or a pharmaceuticallyacceptable composition or dosage form thereof, as described herein.

Compounds, methods, dosage forms, and compositions are provided for thetreatment of a host infected with a HCV virus via administration of aneffective amount of Compound 2. In certain embodiments, Compound 2 isadministered at a dose of at least about 100, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, or 1000 mg. Incertain embodiments, Compound 2 is administered for up to 12 weeks, forup to 10 weeks, for up to 8 weeks, for up to 6 weeks, or for up to 4weeks. In alternative embodiments, Compound 2 is administered for atleast 4 weeks, for at least 6 weeks, for at least 8 weeks, for at least10 weeks, or for at least 12 weeks. In certain embodiments, Compound 2is administered at least once a day or every other day. In certainembodiments, Compound 2 is administered in a dosage form that achieves asteady-state trough plasma level (C_(24,ss)) of metabolite 1-7 betweenapproximately 15-75 ng/mL. In one embodiment, Compound 2 is administeredin a dosage form that achieves a steady-state trough plasma level(C_(24,ss)) of metabolite 1-7 between approximately 20-60 ng/mL. Incertain embodiments, Compound 2 is administered in a dosage form thatachieves an AUC of metabolite 1-7 between approximately 1,200 ng*h/mLand 3,000 ng*h/mL. In one embodiment, Compound 2 is administered in adosage form that achieves an AUC of metabolite 1-7 between approximately1,500 and 2,100 ng*h/mL.

The compounds, compositions, and dosage forms can also be used to treatrelated conditions such as anti-HCV antibody positive and antigenpositive conditions, viral-based chronic liver inflammation, livercancer resulting from advanced hepatitis C (hepatocellular carcinoma(HCC)), cirrhosis, chronic or acute hepatitis C, fulminant hepatitis C,chronic persistent hepatitis C and anti-HCV-based fatigue. The compoundor formulations that include the compounds can also be usedprophylactically to prevent or restrict the progression of clinicalillness in individuals who are anti-HCV antibody- or antigen-positive orwho have been exposed to hepatitis C.

The present invention thus includes the following features:

(a) Compound 2 as described herein;

(b) Prodrugs of Compound 2

(c) Use of Compound 2 in the manufacture of a medicament for treatmentof a hepatitis C virus infection;

(d) Compound 2 for use to treat hepatitis C, optionally in apharmaceutically acceptable carrier;

(e) A method for manufacturing a medicament intended for the therapeuticuse for treating a hepatitis C virus infection, characterized in thatCompound 2, or a pharmaceutically acceptable salt, as described hereinis used in the manufacture;

(e) A pharmaceutical formulation comprising an effective host-treatingamount of Compound 2 with a pharmaceutically acceptable carrier ordiluent;

(f) Processes for the preparation of therapeutic products that containan effective amount of Compound 2;

(g) Solid dosage forms, including those that provide an advantageouspharmacokinetic profile; and

(h) Processes for the manufacture of Compound 2, as described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is an overlay of XRPD diffractograms of samples 1-1 (amorphousCompound 1), 1-2 (crystalline Compound 1), and 1-3 (amorphous Compound2) prior to stability studies for characterization purposes as describedin Example 2 and Example 5. The x-axis is 2Theta measured in degrees andthe y-axis is intensity measured in counts.

FIG. 1B is the HPLC chromatograph of amorphous Compound 1 (sample 1-1)to determine purity as described in Example 2. The purity of the samplewas 98.7%. The x-axis is time measured in minutes and the y-axis isintensity measured in counts.

FIG. 2A is the HPLC chromatograph of crystalline Compound 1 (sample 1-2)to determine purity as described in Example 2. The purity of the samplewas 99.11%. The x-axis is time measured in minutes and the y-axis isintensity measured in counts.

FIG. 2B is a DSC and TGA graph of crystalline Compound 1 (sample 1-2)prior to any stability studies for characterization purposes asdescribed in Example 2. The x-axis is temperature measured in ° C., theleft y-axis heat flow measured in (W/g), and the right y-axis is weightmeasured in percent.

FIG. 3 is an X-ray crystallography image of Compound 1 showing theabsolute stereochemistry as described in Example 2.

FIG. 4A is an overlay of XRPD diffractograms of samples 1-1 (amorphousCompound 1), 1-2 (crystalline Compound 1), and 1-3 (amorphous Compound2) after storing at 25° C. and 60% relative humidity for 14 days asdescribed in Example 2. The x-axis is 2Theta measured in degrees and they-axis is intensity measured in counts.

FIG. 4B is an overlay of XRPD diffractograms of samples 1-4, 1-5, 1-6,1-7, and 1-9 after storing at 25° C. and 60% relative humidity for 7days as described in Example 4. The x-axis is 2Theta measured in degreesand the y-axis is intensity measured in counts.

FIG. 5A is an overlay of XRPD diffractograms of samples 1-4, 1-6, 1-7,and 1-9 after storing at 25° C. and 60% relative humidity for 14 days asdescribed in Example 4. The x-axis is 2Theta measured in degrees and they-axis is intensity measured in counts.

FIG. 5B is the XRPD pattern of amorphous Compound 2 (sample 1-3) asdescribed in Example 5. The x-axis is 2Theta measured in degrees and they-axis is intensity measured in counts.

FIG. 6A is the HPLC chromatograph of amorphous Compound 2 (sample 1-3)to determine purity as described in Example 5. The purity of the samplewas 99.6%. The x-axis is time measured in minutes and the y-axis isintensity measured in counts.

FIG. 6B is a DSC and TGA graph for amorphous Compound 2 (sample 1-3)prior to any stability studies for characterization purposes asdescribed in Example 5. The x-axis is temperature measured in ° C., theleft y-axis heat flow measured in (W/g), and the right y-axis is weightmeasured in percent.

FIG. 7A is an overlay of XRPD diffractograms of crystalline samples(samples 2-2, 2-6, and 2-7) and poorly crystalline samples (samples 2-3,2-4, 2-5, and 2-8) identified from the crystallizations of Compound 2(Example 6). The x-axis is 2Theta measured in degrees and the y-axisintensity measured in counts.

FIG. 7B is an overlay of XRPD diffractograms of amorphous samples(samples 2-9, 2-10, and 2-11) identified from the crystallizations ofCompound 2 (Example 6). The x-axis is 2Theta measured in degrees and they-axis intensity measured in counts.

FIG. 8A is an overlay of XRPD diffractograms of samples (samples 2-2,2-3, 2-4, 2-5, 2-6, 2-7 and 2-8) after 6 days storage at 25° C. and 60%relative humidity (Example 6). The x-axis is 2Theta measured in degreesand the y-axis intensity measured in counts.

FIG. 8B is a DSC and TGA graph for sample 2-2 (Example 6). The x-axis istemperature measured in ° C., the left y-axis heat flow measured in(W/g), and the right y-axis is weight measured in percent. Experimentalprocedures for DSC and TGA collection are given in Example 2.

FIG. 9A is a DSC and TGA graph for sample 2-3 (Example 6). The x-axis istemperature measured in ° C., the left y-axis heat flow measured in(W/g), and the right y-axis is weight measured in percent. Experimentalprocedures for DSC and TGA collection are given in Example 2.

FIG. 9B is a DSC and TGA graph for sample 2-4 (Example 6). The x-axis istemperature measured in ° C., the left y-axis heat flow measured in(W/g), and the right y-axis is weight measured in percent. Experimentalprocedures for DSC and TGA collection are given in Example 2.

FIG. 10A is a DSC and TGA graph for sample 2-5 (Example 6). The x-axisis temperature measured in ° C., the left y-axis heat flow measured in(W/g), and the right y-axis is weight measured in percent. Experimentalprocedures for DSC and TGA collection are given in Example 2.

FIG. 10B is a DSC and TGA graph for sample 2-6 (Example 6). The x-axisis temperature measured in ° C., the left y-axis heat flow measured in(W/g), and the right y-axis is weight measured in percent. Experimentalprocedures for DSC and TGA collection are given in Example 2.

FIG. 11A is a DSC and TGA graph for sample 2-7 (Example 6). The x-axisis temperature measured in ° C., the left y-axis heat flow measured in(W/g), and the right y-axis is weight measured in percent. Experimentalprocedures for DSC and TGA collection are given in Example 2.

FIG. 11B is a DSC and TGA graph for sample 2-8 (Example 6). The x-axisis temperature measured in ° C., the left y-axis heat flow measured in(W/g), and the right y-axis is weight measured in percent. Experimentalprocedures for DSC and TGA collection are given in Example 2.

FIG. 12A is the XRPD pattern of amorphous Compound 4 (sample 3-12) asdiscussed in Example 7. The x-axis is 2Theta measured in degrees and they-axis is intensity measured in counts. No crystallization of a malonatesalt was observed regardless of the solvent used.

FIG. 12B is an overlay of XRPD diffractograms of amorphous samples(samples 3-6, 3-10, 3-11, and 3-12) identified from the attemptedcrystallization of compound 1 with malonate salt (Example 7). The x-axisis 2Theta measured in degrees and the y-axis is intensity measured incounts.

FIG. 13A is the HPLC chromatogram of sample 3-12 from the attemptedcrystallizations of compound 1 with malonate salt as described inExample 7. The sample was 99.2% pure. The x-axis is time measured inminutes and the y-axis is intensity measured in mAu.

FIG. 13B is an overlay of XRPD diffractograms of solid samples obtainedfrom the crystallization using LAG (samples 4-13, 4-12, 4-9, 4-3, and4-1) compared to Compound 1 (sample 1-2) as described in Example 8. Allthe XRDP match the patterns of the crystalline acid counter ion with noadditional peaks. The x-axis is 2Theta measured in degrees and they-axis is intensity measured in counts.

FIG. 14A is an overlay of XRPD diffractograms of samples obtained fromutilizing ethyl acetate as a crystallization solvent (samples 6-13,6-12, 6-11, 6-10, 6-8, 6-7, 6-6, 6-5, 6-4, and 6-2) compared tocrystalline Compound 1 (sample 1-2) as described in Example 10. The XRPDpatterns were generally found to match the Compound 1 pattern with theexception of samples 6-2, 6-4, and 6-5 that exhibit slight differences.The x-axis is 2Theta measured in degrees and the y-axis is intensitymeasured in counts.

FIG. 14B is an overlay of XRPD diffractogram of sample 5-1 following asecond dissolution in MEK and the addition of the antisolventcyclohexane and pamioc acid as described in Example 9. Sample 5-1,crystallized in pamioc acid, was a solid following maturation, but theXRPD pattern matched the pattern of pamioc acid.

FIG. 15A is an overlay of XRPD diffractograms of samples obtained fromutilizing ethyl acetate as a crystallization solvent (samples 6-5, 6-4,and 6-2) compared to crystalline Compound 1 (sample 1-2) as described inExample 10. The XRPD patterns were generally found to match the Compound1 pattern with the exception of samples 6-2, 6-4, and 6-5 that exhibitslight differences. The x-axis is 2Theta measured in degrees and they-axis is intensity measured in counts and labeled with the acid used incrystallization.

FIG. 15B is the XRPD pattern for Compound 2 as described in Example 14.The x-axis is 2Theta measured in degrees and the y-axis is intensitymeasured in counts.

FIG. 16A is a graph of the active TP (metabolite 1-6) concentrationlevels in the livers and hearts of rats, dogs, and monkeys (Example 18).The x-axis is the dosage measured in mg/kg for each species and they-axis is the active TP concentration measured in ng/g.

FIG. 16B is a graph of the active TP (metabolite 1-6) concentrationlevels in the liver and heart of dogs (n=2) measured 4 hours after asingle oral dose of Compound 1 or Compound 2 (Example 19). The x-axis isthe dosage of each compound measured in mg/kg and the y-axis is theactive TP concentration measured in ng/g.

FIG. 17 is the plasma profile of Compound 1 and metabolite 1-7 in ratsgiven a single 500 mg/kg oral dose of Compound 2 (Example 20) measured72 hours post-dose. The x-axis is time measured in hours and the y-axisis plasma concentration measured in ng/mL.

FIG. 18 is the plasma profile of Compound 1 and metabolite 1-7 inmonkeys given single oral doses of 30 mg, 100 mg, or 300 mg of Compound2 (Example 20) measured 72 hours post-dose. The x-axis is time measuredin hours and the y-axis is plasma concentration measured in ng/mL.

FIG. 19 is a graph of EC₉₅ measured in nM of sofosbuvir and Compound 1against HCV clinical isolates. EC₉₅ values for Compound 1 are 7-33 timeslower than sofosbuvir (Example 22). The x-axis is labeled with thegenotype and the y-axis is EC₉₅ measured in nM.

FIG. 20 is a graph of EC₅₀ measured in nM of sofosbuvir and Compound 1against laboratory strains of HCV Genotypes 1a, 1b, 2a, 3a, 4a, and 5a.Compound 1 is approximately 6-11 times more potent than sofosbuvir inGenotypes 1-5 (Example 22). The x-axis is labeled with the genotype andthe y-axis is EC₅₀ measured in nM.

FIG. 21 is a graph of the mean plasma concentration-time profile ofCompound 1 following the administration of a single dose of Compound 2in all cohorts of Part B of the study as described in Example 24.Compound 1 was quickly absorbed and rapidly metabolized withinapproximately 8 hours in all cohorts from Part B. The x-axis is the timemeasured in hours and the y-axis is the geometric mean plasmaconcentration measured in ng/mL.

FIG. 22 is a graph of the mean plasma concentration-time profile ofmetabolite 1-7 following the administration of a single dose of Compound2 in all cohorts of Part B of the study as described in Example 24.Metabolite 1-7 exhibited sustained plasma concentration in all cohortsfrom Part B. The x-axis is the time measured in hours and the y-axis isthe geometric mean plasma concentration measured in ng/mL.

FIG. 23A is an individual pharmacokinetic/pharmacodynamic analysis of asubject enrolled in the 1b cohort as described in Example 24. The graphshows plasma metabolite 1-7 exposure and HCV RNA reduction levels. Thedashed line represents the minimum concentration of metabolite 1-7required to sustain a viral response greater than the EC₉₅ value againstGT1b. The x-axis is time measured in hours. The left y-axis ismetabolite 1-7 plasma concentration measured in ng/mL and the righty-axis is the HCV RNA reduction measured in log₁₀ IU/mL.

FIG. 23B is an individual pharmacokinetic/pharmacodynamic analysis of asubject enrolled in the 1b cohort as described in Example 24. The graphshows plasma metabolite 1-7 exposure and HCV RNA reduction levels. Thedashed line represents the minimum concentration of metabolite 1-7required to sustain a viral response greater than the EC₉₅ value againstGT1b. The x-axis is time measured in hours. The left y-axis ismetabolite 1-7 plasma concentration measured in ng/mL and the righty-axis is the HCV RNA reduction measured in log₁₀ IU/mL.

FIG. 23C is an individual pharmacokinetic/pharmacodynamic analysis of asubject enrolled in the 1b cohort as described in Example 24. The graphshows plasma metabolite 1-7 exposure and HCV RNA reduction levels. Thedashed line represents the minimum concentration of metabolite 1-7required to sustain a viral response greater than the EC₉₅ value againstGT1b. The x-axis is time measured in hours. The left y-axis ismetabolite 1-7 plasma concentration measured in ng/mL and the righty-axis is the HCV RNA reduction measured in log₁₀ IU/mL.

FIG. 23D is an individual pharmacokinetic/pharmacodynamic analysis of asubject enrolled in the 3b cohort as described in Example 24. Each graphshows plasma metabolite 1-7 exposure and HCV RNA reduction levels. Thedashed line represents the minimum concentration of metabolite 1-7required to sustain a viral response greater than the EC₉₅ value againstGT1b. The x-axis is time measured in hours. The left y-axis ismetabolite 1-7 plasma concentration measured in ng/mL and the righty-axis is the HCV RNA reduction measured in log₁₀ IU/mL.

FIG. 23E is an individual pharmacokinetic/pharmacodynamic analysis of asubject enrolled in the 3b cohort as described in Example 24. Each graphshows plasma metabolite 1-7 exposure and HCV RNA reduction levels. Thedashed line represents the minimum concentration of metabolite 1-7required to sustain a viral response greater than the EC₉₅ value againstGT1b. The x-axis is time measured in hours. The left y-axis ismetabolite 1-7 plasma concentration measured in ng/mL and the righty-axis is the HCV RNA reduction measured in log₁₀ IU/mL.

FIG. 23F is an individual pharmacokinetic/pharmacodynamic analysis of asubject enrolled in the 3b cohort as described in Example 24. Each graphshows plasma metabolite 1-7 exposure and HCV RNA reduction levels. Thedashed line represents the minimum concentration of metabolite 1-7required to sustain a viral response greater than the EC₉₅ value againstGT1b. The x-axis is time measured in hours. The left y-axis ismetabolite 1-7 plasma concentration measured in ng/mL and the righty-axis is the HCV RNA reduction measured in log₁₀ IU/mL.

FIG. 24 is a graph of the EC₉₅ values of Compound 1 and sofosbuviragainst clinical isolates of GT1, GT2, GT3, and GT4 HCV-infectedpatients. The dashed horizontal line (

) represents the steady-state trough concentration (C_(24,ss)) ofsofosbuvir nucleoside following a dose of 400 mg QD of sofosbuvir. Thefull horizontal line (

)represents the steady-state trough concentration (C_(24,ss)) ofmetabolite 1-7 following 600 mg of Compound 2 (equivalent to 550 mg ofCompound 1). The dotted horizontal line (

)represents the steady-state trough concentration (C_(24,ss)) ofmetabolite 1-7 following 450 mg of Compound 2 (equivalent to 400 mg ofCompound 1). As discussed in Example 25, the predicted steady-statetrough plasma level (C_(24,ss)) of metabolite 1-7 following 600 mg and450 mg of Compound 2 exceeds the in vitro EC₉₅ of Compound 1 against alltested clinical isolates. The steady state trough plasma level(C_(24,ss)) of sofosbuvir only exceeds the EC₉₅ at GT2 clinicalisolates. The x-axis is labeled with the clinical isolates and the tableunder the x-axis lists the EC₉₅ values for Compound 1 and sofosbuvir.The y-axis is the EC₉₅ against the clinical isolates measured in ng/mL.EC₉₅ is expressed as nucleoside equivalent. Sofosbuvir and Compound 2were administered daily (QD).

FIG. 25 is a flow diagram showing the manufacturing process of 50 mg and100 mg tablets of Compound 2 as described in Example 26. In step 1,microcrystalline cellulose, Compound 2, lactose monohydrate, andcroscarmellose sodium are filtered through a 600 μM screen. In step 2,the contents from step 1 are loaded into a V-blender and mixed for 5minutes at 25 rpm. In step 3, magnesium stearate is filtered through a600 μM screen. In step 4, magnesium stearate is loaded into theV-blender containing the contents from step 2 (microcrystallinecellulose, Compound 2, lactose monohydrate, and croscarmellose sodium)and mixed for 2 minutes at 25 rpm. The common blend is then divided forthe production of 50 mg tablets and 100 mg tablets. To produce 50 mgtablets, the blend from step 4 is compressed with 6 mm round standardconcave tooling. To produce 100 mg tablets, the blend from step 4 iscompressed with 8 mm round standard concave tooling. The tablets arethen packaged into HDPE bottles induction-sealed with PP caps withdesiccant.

FIG. 26 is the hemi-sulfate salt that exhibits advantageouspharmacological properties over its corresponding free base for thetreatment of an HCV virus.

DETAILED DESCRIPTION OF THE INVENTION

The invention disclosed herein is a compound, method, composition, andsolid dosage form for the treatment of infections in or exposure tohumans and other host animals of the HCV virus that includes theadministration of an effective amount of the hemi-sulfate salt ofisopropyl((S)-(((2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate(Compound 2) as described herein, optionally in a pharmaceuticallyacceptable carrier. In one embodiment, Compound 2 is an amorphous solid.In yet another embodiment, Compound 2 is a crystalline solid.

The compound, compositions, and dosage forms can also be used to treatconditions related to or occurring as a result of an HCV viral exposure.For example, the active compound can be used to treat HCV antibodypositive- and HCV antigen-positive conditions, viral-based chronic liverinflammation, liver cancer resulting from advanced hepatitis C (e.g,hepatocellular carcinoma), cirrhosis, acute hepatitis C, fulminanthepatitis C, chronic persistent hepatitis C, and anti-HCV-based fatigue.

The active compounds and compositions can also be used to treat therange of HCV genotypes. At least six distinct genotypes of HCV, each ofwhich have multiple subtypes, have been identified globally. Genotypes1-3 are prevalent worldwide, and Genotypes 4, 5, and 6 are more limitedgeographically. Genotype 4 is common in the Middle East and Africa.Genotype 5 is mostly found in South Africa. Genotype 6 predominatelyexists in Southeast Asia. Although the most common genotype in theUnited States is Genotype 1, defining the genotype and subtype canassist in treatment type and duration. For example, different genotypesrespond differently to different medications and optimal treatment timesvary depending on the genotype infection. Within genotypes, subtypes,such as Genotype 1a and Genotype 1b, respond differently to treatment aswell. Infection with one type of genotype does not preclude a laterinfection with a different genotype.

As described in Example 22, Compound 2 is active against the range ofHCV genotypes, including Genotypes 1-5. In one embodiment, Compound 2 isused to treat HCV Genotype 1, HCV Genotype 2, HCV Genotype 3, HCVGenotype 4, HCV Genotype 5, or HCV Genotype 6. In one embodiment,Compound 2 is used to treat HCV Genotype 1a. In one embodiment, Compound2 is used to treat HCV Genotype 1b. In one embodiment, Compound 2 isused to treat HCV Genotype 2a. In one embodiment, Compound 2 is used totreat HCV Genotype 2b. In one embodiment, Compound 2 is used to treatHCV Genotype 3a. In one embodiment, Compound 2 is used to treat HCVGenotype 4a. In one embodiment, Compound 2 is used to treat HCV Genotype4d.

In one embodiment, Compound 1 or Compound 2 is used to treat HCVGenotype 5a. In one embodiment, Compound 1 or Compound 2 is used totreat HCV Genotype 6a. In one embodiment, Compound 1 or Compound 2 isused to treat HCV Genotype 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 6l,6m, 6n, 6o, 6p, 6q, 6r, 6s, 6t, or 6u.

As discussed in Example 25 and shown in FIG. 24, the predictedsteady-state trough concentration (C_(24,ss)) of metabolite 1-7following a dose of 450 mg (400 mg free base) and a dose of 600 mg (550mg free base) of Compound 2 is approximately 40 ng/mL to 50 ng/mL. ThisC_(24,ss) level exceeded the EC₉₅ of Compound 1 at HCV Genotypes 1a, 1b,2a, 2b, 3a, 4a, and 4d. This data confirms that Compound 2 haspotent-pan genotypic activity. This is surprising because Compound 2achieves a smaller steady-state trough concentration (C_(24,ss)) thanthe steady-state trough concentration (C_(24,ss)) of the nucleosidemetabolite of sofosbuvir following equivalent sofosbuvir dosing. Thesteady-state trough concentration (C_(24,ss)) of the correspondingnucleoside metabolite of sofosbuvir is approximately 100 ng/mL, but thislevel only exceeds the EC₉₅ of sofosbuvir against GT2 clinical isolates(FIG. 24). Compound 2 is more potent than sofosbuvir against GT1, GT2,GT3, and GT4, and therefore allows a dosage form that delivers a smallersteady-state trough concentration of its metabolite which is nonethelessefficacious against all tested genotypes of HCV. In one embodiment, adosage form of Compound 2 is delivered that achieves a metabolite 1-7steady-state trough concentration (C_(24,ss)) between approximately15-75 ng/mL. In one embodiment, a dosage form of Compound 2 is deliveredthat achieves a metabolite 1-7 steady-state trough concentration(C_(24,ss)) between approximately 20-60 ng/mL, 20-50 ng/mL, or 20-40ng/mL.

In one embodiment, the compound, formulations, or solid dosage formsthat include the compound can also be used prophylactically to preventor retard the progression of clinical illness in individuals who are HCVantibody- or HCV antigen-positive or who have been exposed to hepatitisC.

In particular, it has been discovered that Compound 2 is active againstHCV and exhibits superior drug-like and pharmacological propertiescompared to its free base (Compound 1). Surprisingly, Compound 2 is morebioavailable and achieves a higher AUC than Compound 1 (Example 19) andCompound 2 is more selective for the target organ, the liver, thanCompound 1 (Example 19).

Compound 2 is also advantageous over Compound 1 in terms of solubilityand chemical stability. This is surprising because the mono-sulfate saltofisopropyl((S)-(((2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (Compound 3) isunstable and exhibits the appearance of a sticky gum, while Compound 2,the hemi-sulfate salt, is a stable white solid. The hemisulfate salt,both as a solid and in a solid dosage form, is very stable over 9 monthsand is not hydroscopic.

Despite the volume of antiviral nucleoside literature and patentfilings, Compound 2 has not been specifically disclosed.

Compound 2 has S-stereochemistry at the phosphorus atom which has beenconfirmed with X-ray crystallography (FIG. 3, Example 2). In alternativeembodiments, Compound 2 can be used in the form of any desired ratio ofphosphorus R- and S-enantiomers, including up to pure enantiomers. Insome embodiments, Compound 2 is used in a form that is at least 90% freeof the opposite enantiomer, and can be at least 98%, 99%, or even 100%free of the opposite enantiomer. Unless described otherwise, anenantiomerically enriched Compound 2 is at least 90% free of theopposite enantiomer. In addition, in an alternative embodiment, theamino acid of the phosphoramidate can be in the D- or L-configuration,or a mixture thereof, including a racemic mixture.

Unless otherwise specified, the compounds described herein are providedin the β-D-configuration. In an alternative embodiment, the compoundscan be provided in a β-L-configuration. Likewise, any substituent groupthat exhibits chirality can be provided in racemic, enantiomeric,diastereomeric form, or any mixture thereof. Where a phosphoramidateexhibits chirality, it can be provided as an R or S chiral phosphorusderivative or a mixture thereof, including a racemic mixture. All of thecombinations of these stereo configurations are alternative embodimentsin the invention described herein. In another embodiment, at least oneof the hydrogens of Compound 2 (the nucleotide or the hemi-sulfate salt)can be replaced with deuterium.

These alternative configurations include, but are not limited to,

I. Hemi-Sulfate Salt ofisopropyl((S)-(((2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate(Compound 2)

The active compound of the invention is Compound 2, which can beprovided in a pharmaceutically acceptable composition or solid dosageform thereof. In one embodiment, Compound 2 is an amorphous solid. Inyet a further embodiment, Compound 2 is a crystalline solid.

Synthesis of Compound 2

The present invention further includes a non-limiting illustrativeprocess for the preparation of Compound 2 that includes

(i) a first step of dissolving Compound 1 in an organic solvent, forexample, acetone, ethyl acetate, methanol, acetonitrile, or ether, orthe like, in a flask or container;

(ii) charging a second flask or container with a second organic solvent,which may be the same as or different from the organic solvent in step(i), optionally cooling the second solvent to 0-10 degrees C., andadding dropwise H₂SO₄ to the second organic solvent to create aH₂SO₄/organic solvent mixture; and wherein the solvent for example maybe methanol;

(iii) adding dropwise the H₂SO₄/solvent mixture at a molar ratio of0.5/1.0 from step (ii) to the solution of Compound 1 of step (i) atambient or slightly increased or decreased temperature (for example23-35 degrees C.);

(iv) stirring the reaction of step (iii) until precipitate of Compound 2is formed, for example at ambient or slightly increased or decreasedtemperature;

(v) optionally filtering the resulting precipitate from step (iv) andwashing with an organic solvent; and

(vi) optionally drying the resulting Compound 2 in a vacuum, optionallyat elevated a temperature, for example, 55, 56, 57, 58, 59, or 60° C.

In certain embodiments, step (i) above is carried out in acetone.Further, the second organic solvent in step (ii) may be for examplemethanol and the mixture of organic solvents in step (v) ismethanol/acetone.

In one embodiment, Compound 1 is dissolved in ethyl acetate in step (i).In one embodiment, Compound 1 is dissolved in tetrahydrofuran in step(i). In one embodiment, Compound 1 is dissolved in acetonitrile in step(i). In an additional embodiment, Compound 1 is dissolved indimethylformamide in step (i).

In one embodiment, the second organic solvent in step (ii) is ethanol.In one embodiment, the second organic solvent in step (ii) isisopropanol. In one embodiment, the second organic solvent in step (ii)is n-butanol.

In one embodiment, a mixture of solvents are used for washing in step(v), for example, ethanol/acetone. In one embodiment, the mixture ofsolvent for washing in step (v) is isopropanol/acetone. In oneembodiment, the mixture of solvent for washing in step (v) isn-butanol/acetone. In one embodiment, the mixture of solvent for washingin step (v) is ethanol/ethyl acetate. In one embodiment, the mixture ofsolvent for washing in step (v) is isopropanol/ethyl acetate. In oneembodiment, the mixture of solvent for washing in step (v) isn-butanol/ethyl acetate. In one embodiment, the mixture of solvent forwashing in step (v) is ethanol/tetrahydrofuran. In one embodiment, themixture of solvent for washing in step (v) isisopropanol/tetrahydrofuran. In one embodiment, the mixture of solventfor washing in step (v) is n-butanol/tetrahydrofuran. In one embodiment,the mixture of solvent for washing in step (v) is ethanol/acetonitrile.In one embodiment, the mixture of solvent for washing in step (v) isisopropanol/acetonitrile. In one embodiment, the mixture of solvent forwashing in step (v) is n-butanol/acetonitrile. In one embodiment, themixture of solvent for washing in step (v) is ethanol/dimethylformamide.In one embodiment, the mixture of solvent for washing in step (v) isisopropanol/dimethylformamide. In one embodiment, the mixture of solventfor washing in step (v) is n-butanol/dimethylformamide.

II. Metabolism ofIsopropyl((S)-(((2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate(Compound 2)

The metabolism of Compound 1 and Compound 2 involves the production of a5′-monophosphate and the subsequent anabolism of theN⁶-methyl-2,6-diaminopurine base (1-3) to generate ((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methyldihydrogen phosphate (1-4) as the 5′-monophosphate. The monophosphate isthen further anabolized to the active triphosphate species: the5′-triphosphate (1-6). The 5′-triphosphate can be further metabolized togenerate2-amino-9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (1-7). Alternatively,5′-monophophate 1-2 can be metabolized to generate the purine base 1-8.The metabolic pathway forisopropyl((S)-(((2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninateis illustrated in Scheme 1 (shown above).

III. Additional Salts of Compound 1

In alternative embodiments, the present invention provides Compound 1 asan oxalate salt (Compound 4) or an HCl salt (Compound 5).

Both the 1:1 oxalate salt and the 1:1 HCl salt form solids withreasonable properties for solid dosage forms for the treatment of a hostsuch as a human with hepatitis C. However, the oxalate salt may be lessdesired, and perhaps not suitable, if the patient is susceptible tokidney stones. The HCl salt is more hydroscopic than the hemisulfatesalt. Thus, the hemisulfate salt remains the most desired salt form ofCompound 1 with unexpected properties.

IV. Definitions

The term “D-configuration” as used in the context of the presentinvention refers to the principle configuration which mimics the naturalconfiguration of sugar moieties as opposed to the unnatural occurringnucleosides or “L” configuration. The term “β” or “βanomer” is used withreference to nucleoside analogs in which the nucleoside base isconfigured (disposed) above the plane of the furanose moiety in thenucleoside analog.

The terms “coadminister” and “coadministration” or combination therapyare used to describe the administration of Compound 2 according to thepresent invention in combination with at least one other active agent,for example where appropriate at least one additional anti-HCV agent.The timing of the coadministration is best determined by the medicalspecialist treating the patient. It is sometimes preferred that theagents be administered at the same time. Alternatively, the drugsselected for combination therapy may be administered at different timesto the patient. Of course, when more than one viral or other infectionor other condition is present, the present compounds may be combinedwith other agents to treat that other infection or condition asrequired.

The term “host”, as used herein, refers to a unicellular ormulticellular organism in which a HCV virus can replicate, includingcell lines and animals, and typically a human. The term hostspecifically refers to infected cells, cells transfected with all orpart of a HCV genome, and animals, in particular, primates (includingchimpanzees) and humans. In most animal applications of the presentinvention, the host is a human patient. Veterinary applications, incertain indications, however, are clearly anticipated by the presentinvention (such as chimpanzees). The host can be for example, bovine,equine, avian, canine, feline, etc.

Isotopic Substitution

The present invention includes compounds and the use of compound 2 withdesired isotopic substitutions of atoms at amounts above the naturalabundance of the isotope, i.e., enriched. Isotopes are atoms having thesame atomic number but different mass numbers, i.e., the same number ofprotons but a different number of neutrons. By way of general exampleand without limitation, isotopes of hydrogen, for example, deuterium(²H) and tritium (³H) may be used anywhere in described structures.Alternatively or in addition, isotopes of carbon, e.g., ¹³C and ¹⁴C, maybe used. A preferred isotopic substitution is deuterium for hydrogen atone or more locations on the molecule to improve the performance of thedrug. The deuterium can be bound in a location of bond breakage duringmetabolism (an α-deuterium kinetic isotope effect) or next to or nearthe site of bond breakage (a β-deuterium kinetic isotope effect).Achillion Pharmaceuticals, Inc. (WO/2014/169278 and WO/2014/169280)describes deuteration of nucleotides to improve their pharmacokinetic orpharmacodynamic, including at the 5-position of the molecule.

Substitution with isotopes such as deuterium can afford certaintherapeutic advantages resulting from greater metabolic stability, suchas, for example, increased in vivo half-life or reduced dosagerequirements. Substitution of deuterium for hydrogen at a site ofmetabolic break-down can reduce the rate of or eliminate the metabolismat that bond. At any position of the compound that a hydrogen atom maybe present, the hydrogen atom can be any isotope of hydrogen, includingprotium (¹H), deuterium (²H) and tritium (³H). Thus, reference herein toa compound encompasses all potential isotopic forms unless the contextclearly dictates otherwise.

The term “isotopically-labeled” analog refers to an analog that is a“deuterated analog”, a “¹³C-labeled analog,” or a“deuterated/¹³C-labeled analog.” The term “deuterated analog” means acompound described herein, whereby a H-isotope, i.e., hydrogen/protium(¹H), is substituted by a H-isotope, i.e., deuterium (²H). Deuteriumsubstitution can be partial or complete. Partial deuterium substitutionmeans that at least one hydrogen is substituted by at least onedeuterium. In certain embodiments, the isotope is 90, 95 or 99% or moreenriched in an isotope at any location of interest. In some embodimentsit is deuterium that is 90, 95 or 99% enriched at a desired location.Unless indicated to the contrary, the deuteration is at least 80% at theselected location. Deuteration of the nucleoside can occur at anyreplaceable hydrogen that provides the desired results.

V. Methods of Treatment or Prophylaxis

Treatment, as used herein, refers to the administration of Compound 2 toa host, for example a human that is or may become infected with a HCVvirus.

The term “prophylactic” or preventative, when used, refers to theadministration of Compound 2 to prevent or reduce the likelihood of anoccurrence of the viral disorder. The present invention includes bothtreatment and prophylactic or preventative therapies. In one embodiment,Compound 2 is administered to a host who has been exposed to and thus isat risk of infection by a hepatitis C virus infection.

The invention is directed to a method of treatment or prophylaxis of ahepatitis C virus, including drug resistant and multidrug resistantforms of HCV and related disease states, conditions, or complications ofan HCV infection, including cirrhosis and related hepatotoxicities, aswell as other conditions that are secondary to a HCV infection, such asweakness, loss of appetite, weight loss, breast enlargement (especiallyin men), rash (especially on the palms), difficulty with clotting ofblood, spider-like blood vessels on the skin, confusion, coma(encephalopathy), buildup of fluid in the abdominal cavity (ascites),esophageal varices, portal hypertension, kidney failure, enlargedspleen, decrease in blood cells, anemia, thrombocytopenia, jaundice, andhepatocellular cancer, among others. The method comprises administeringto a host in need thereof, typically a human, with an effective amountof Compound 2 as described herein, optionally in combination with atleast one additional bioactive agent, for example, an additionalanti-HCV agent, further in combination with a pharmaceuticallyacceptable carrier additive and/or excipient.

In yet another aspect, the present invention is a method for preventionor prophylaxis of an HCV infection or a disease state or related orfollow-on disease state, condition or complication of an HCV infection,including cirrhosis and related hepatotoxicities, weakness, loss ofappetite, weight loss, breast enlargement (especially in men), rash(especially on the palms), difficulty with clotting of blood,spider-like blood vessels on the skin, confusion, coma (encephalopathy),buildup of fluid in the abdominal cavity (ascites), esophageal varices,portal hypertension, kidney failure, enlarged spleen, decrease in bloodcells, anemia, thrombocytopenia, jaundice, and hepatocellular (liver)cancer, among others, said method comprising administering to a patientat risk with an effective amount Compound 2 as described above incombination with a pharmaceutically acceptable carrier, additive, orexcipient, optionally in combination with another anti-HCV agent. Inanother embodiment, the active compounds of the invention can beadministered to a patient after a hepatitis-related livertransplantation to protect the new organ.

In an alternative embodiment, Compound 2 is provided as the hemisulfatesalt of a phosphoramidate of Compound 1 other than the specificphosphoramidate described in the compound illustration. A wide range ofphosphoramidates are known to those skilled in the art that includevarious esters and phospho-esters, any combination of which can be usedto provide an active compound as described herein in the form of ahemisulfate salt.

VI. Pharmaceutical Compositions and Dosage Forms

In an aspect of the invention, pharmaceutical compositions according tothe present invention comprise an anti-HCV virus effective amount ofCompound 2 as described herein, optionally in combination with apharmaceutically acceptable carrier, additive, or excipient, furtheroptionally in combination or alternation with at least one other activecompound. In one embodiment, the invention includes a solid dosage formof Compound 2 in a pharmaceutically acceptable carrier.

In an aspect of the invention, pharmaceutical compositions according tothe present invention comprise an anti-HCV effective amount of Compound2 described herein, optionally in combination with a pharmaceuticallyacceptable carrier, additive, or excipient, further optionally incombination with at least one other antiviral agent, such as an anti-HCVagent.

The invention includes pharmaceutical compositions that include aneffective amount to treat a hepatitis C virus infection of Compound 2 ofthe present invention or prodrug, in a pharmaceutically acceptablecarrier or excipient. In an alternative embodiment, the inventionincludes pharmaceutical compositions that include an effective amount toprevent a hepatitis C virus infection of Compound 2 of the presentinvention or prodrug, in a pharmaceutically acceptable carrier orexcipient.

One of ordinary skill in the art will recognize that a therapeuticallyeffective amount will vary with the infection or condition to betreated, its severity, the treatment regimen to be employed, thepharmacokinetic of the agent used, as well as the patient or subject(animal or human) to be treated, and such therapeutic amount can bedetermined by the attending physician or specialist.

Compound 2 according to the present invention can be formulated in amixture with a pharmaceutically acceptable carrier. In general, it ispreferable to administer the pharmaceutical composition inorally-administrable form, an in particular, a solid dosage form such asa pill or tablet. Certain formulations may be administered via aparenteral, intravenous, intramuscular, topical, transdermal, buccal,subcutaneous, suppository, or other route, including intranasal spray.Intravenous and intramuscular formulations are often administered insterile saline. One of ordinary skill in the art may modify theformulations to render them more soluble in water or another vehicle,for example, this can be easily accomplished by minor modifications(salt formulation, esterification, etc.) that are well within theordinary skill in the art. It is also well within the routineers' skillto modify the route of administration and dosage regimen of Compound 2in order to manage the pharmacokinetic of the present compounds formaximum beneficial effect in patients, as described in more detailherein.

In certain pharmaceutical dosage forms, the prodrug form of thecompounds, especially including acylated (acetylated or other), andether (alkyl and related) derivatives, phosphate esters,thiophosphoramidates, phosphoramidates, and various salt forms of thepresent compounds, may be used to achieve the desired effect. One ofordinary skill in the art will recognize how to readily modify thepresent compounds to prodrug forms to facilitate delivery of activecompounds to a targeted site within the host organism or patient. Theperson of ordinary skill in the art also will take advantage offavorable pharmacokinetic parameters of the prodrug forms, whereapplicable, in delivering the present compounds to a targeted sitewithin the host organism or patient to maximize the intended effect ofthe compound.

The amount of Compound 2 included within the therapeutically activeformulation according to the present invention is an effective amount toachieve the desired outcome according to the present invention, forexample, for treating the HCV infection, reducing the likelihood of aHCV infection or the inhibition, reduction, and/or abolition of HCV orits secondary effects, including disease states, conditions, and/orcomplications which occur secondary to HCV. In general, atherapeutically effective amount of the present compound in apharmaceutical dosage form may range from about 0.001 mg/kg to about 100mg/kg per day or more, more often, slightly less than about 0.1 mg/kg tomore than about 25 mg/kg per day of the patient or considerably more,depending upon the compound used, the condition or infection treated andthe route of administration. Compound 2 is often administered in amountsranging from about 0.1 mg/kg to about 15 mg/kg per day of the patient,depending upon the pharmacokinetic of the agent in the patient. Thisdosage range generally produces effective blood level concentrations ofactive compound which may range from about 0.001 to about 100, about0.05 to about 100 micrograms/cc of blood in the patient.

Often, to treat, prevent or delay the onset of these infections and/orto reduce the likelihood of an HCV virus infection, or a secondarydisease state, condition or complication of HCV, Compound 2 will beadministered in a solid dosage form in an amount ranging from about 250micrograms up to about 800 milligrams or more at least once a day, forexample, at least about 5, 10, 20, 25, 50, 75, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 milligrams or more,once, twice, three, or up to four times a day according to the directionof the healthcare provider. Compound 2 often administered orally, butmay be administered parenterally, topically, or in suppository form, aswell as intranasally, as a nasal spray or as otherwise described herein.More generally, Compound 2 can be administered in a tablet, capsule,injection, intravenous formulation, suspension, liquid, emulsion,implant, particle, sphere, cream, ointment, suppository, inhalable form,transdermal form, buccal, sublingual, topical, gel, mucosal, and thelike.

When a dosage form herein refers to a milligram weight dose, it refersto the amount of Compound 2 (i.e., the weight of the hemi-sulfate salt)unless otherwise specified to the contrary.

In certain embodiments, the pharmaceutical composition is in a dosageform that contains from about 1 mg to about 2000 mg, from about 10 mg toabout 1000 mg, from about 100 mg to about 800 mg, from about 200 mg toabout 600 mg, from about 300 mg to about 500 mg, or from about 400 mg toabout 450 mg of Compound 2 in a unit dosage form. In certainembodiments, the pharmaceutical composition is in a dosage form, forexample in a solid dosage form, that contains up to about 10, about 50,about 100, about 125, about 150, about 175, about 200, about 225, about250, about 275, about 300, about 325, about 350, about 375, about 400,about 425, about 450, about 475, about 500, about 525, about 550, about575, about 600, about 625, about 650, about 675, about 700, about 725,about 750, about 775, about 800, about 825, about 850, about 875, about900, about 925, about 950, about 975, or about 1000 mg or more ofCompound 2 in a unit dosage form. In one embodiment, Compound 2 isadministered in a dosage form that delivers at least about 300 mg. Inone embodiment, Compound 2 is administered in a dosage form thatdelivers at least about 400 mg. In one embodiment, Compound 2 isadministered in a dosage form that delivers at least about 500 mg. Inone embodiment, Compound 2 is administered in a dosage form thatdelivers at least about 600 mg. In one embodiment, Compound 2 isadministered in a dosage form that delivers at least about 700 mg. Inone embodiment, Compound 2 is administered in a dosage form thatdelivers at least about 800 mg. In certain embodiments, Compound 2 isadministered at least once a day for up to 12 weeks. In certainembodiments, Compound 2 is administered at least once a day for up to 10weeks. In certain embodiments, Compound 2 is administered at least oncea day for up to 8 weeks. In certain embodiments, Compound 2 isadministered at least once a day for up to 6 weeks. In certainembodiments, Compound 2 is administered at least once a day for up to 4weeks. In certain embodiments, Compound 2 is administered at least oncea day for at least 4 weeks. In certain embodiments, Compound 2 isadministered at least once a day for at least 6 weeks. In certainembodiments, Compound 2 is administered at least once a day for at least8 weeks. In certain embodiments, Compound 2 is administered at leastonce a day for at least 10 weeks. In certain embodiments, Compound 2 isadministered at least once a day for at least 12 weeks. In certainembodiments, Compound 2 is administered at least every other day for upto 12 weeks, up to 10 weeks, up to 8 weeks, up to 6 weeks, or up to 4weeks. In certain embodiments, Compound 2 is administered at least everyother day for at least 4 weeks, at least 6 weeks, at least 8 weeks, atleast 10 weeks, or at least 12 weeks. In one embodiment, at least about600 mg of Compound 2 is administered at least once a day for up to 6weeks. In one embodiment, at least about 500 mg of Compound 2 isadministered at least once a day for up to 6 weeks. In one embodiment,at least about 400 mg of Compound 2 is administered at least once a dayfor up to 6 weeks. In one embodiment, at least 300 mg of Compound 2 isadministered at least once a day for up to 6 weeks. In one embodiment,at least 200 mg of Compound 2 is administered at least once a day for upto 6 weeks. In one embodiment, at least 100 mg of Compound 2 isadministered at least once a day for up to 6 weeks.

Metabolite 1-6 is the active triphosphate of Compound 2, but metabolite1-6 is not measurable in plasma. A surrogate for metabolite 1-6 ismetabolite 1-7. Metabolite 1-7 is a nucleoside metabolite measurable inplasma and is therefore an indication of the intracellularconcentrations of metabolite 1-6. For maximum HCV antiviral activity, adosage form of Compound 2 must achieve a metabolite 1-7 steady-statetrough concentration (C_(24,ss)) that exceeds the EC₉₅ value of Compound2. As shown in FIG. 24, the EC₉₅ of Compound 1 against clinical isolatesof GT1, GT2, GT3, and GT4 is less than 25 ng/mL (Compound 1 EC₉₅ andCompound 2 EC₉₅ values are the same). In one embodiment, a dosage formof Compound 2 is delivered that achieves a steady-state troughconcentration (C_(24,ss)) of metabolite 1-7 that is betweenapproximately 15 to 75 ng/mL. In one embodiment, a dosage form ofCompound 2 is delivered that achieves a steady-state troughconcentration (C_(24,ss)) of metabolite 1-7 that is betweenapproximately 20 to 60 ng/mL. In one embodiment, a dosage form ofCompound 2 is delivered that achieves a steady-state troughconcentration (C_(24,ss)) of metabolite 1-7 that is betweenapproximately 30 to 60 ng/mL. In one embodiment, a dosage form ofCompound 2 is delivered that achieves a steady-state troughconcentration (C_(24,ss)) of metabolite 1-7 that is betweenapproximately 20 to 50 ng/mL. In one embodiment, a dosage form ofCompound 2 is delivered that achieves a steady-state troughconcentration (C_(24,ss)) of metabolite 1-7 that is betweenapproximately 30 to 50 ng/mL. In one embodiment, a dosage form ofCompound 2 is delivered that achieves a steady-state troughconcentration (C_(24,ss)) of metabolite 1-7 that is betweenapproximately 20 to 45 ng/mL. In one embodiment, a dosage form ofCompound 2 is delivered that achieves a steady-state troughconcentration (C_(24,ss)) of metabolite 1-7 that is betweenapproximately 20 to 30 ng/mL. In one embodiment, a dosage form ofCompound 2 is delivered that achieves a steady-state troughconcentration (C_(24,ss)) of metabolite 1-7 that is betweenapproximately 20 to 35 ng/mL. In one embodiment, a dosage form ofCompound 2 is delivered that achieves a steady-state troughconcentration (C_(24,ss)) of metabolite 1-7 that is betweenapproximately 20 to 25 ng/mL. Approximate dosage forms are ±10% of thesteady-state trough concentration.

In one embodiment, Compound 2 is dosed at an amount that achieves ametabolite 1-7 AUC (area under the curve) of between approximately 1,200and 3,000 ng/mL. In one embodiment, Compound 2 is dosed at an amountthat achieves a metabolite 1-7 AUC of between approximately 1,500 and3,000 ng/mL. In one embodiment, Compound 2 is dosed at an amount thatachieves a metabolite 1-7 AUC of between approximately 1,800 and 3,000ng/mL. In one embodiment, Compound 2 is dosed at an amount that achievesa metabolite 1-7 AUC of between approximately 2,100 and 3,000 ng/mL. Ina preferred embodiment, Compound 2 is dosed at amount that achieves ametabolite 1-7 AUC of approximately 2,200 ng*h/mL. Approximate dosageforms are ±10% of the AUC.

In the case of the co-administration of Compound 2 in combination withanother anti-HCV compound as otherwise described herein, the amount ofCompound 2 according to the present invention to be administered inranges from about 0.01 mg/kg of the patient to about 800 mg/kg or moreof the patient or considerably more, depending upon the second agent tobe co-administered and its potency against the virus, the condition ofthe patient and severity of the disease or infection to be treated andthe route of administration. The other anti-HCV agent may for example beadministered in amounts ranging from about 0.01 mg/kg to about 800mg/kg. Examples of dosage amounts of the second active agent are amountsranging from about 250 micrograms up to about 750 mg or more at leastonce a day, for example, at least about 5, 10, 20, 25, 50, 75, 100, 150,200, 250, 300, 350, 400, 450, 500, 600, 700, or 800 milligrams or more,up to four times a day. In certain preferred embodiments, Compound 2 maybe often administered in an amount ranging from about 0.5 mg/kg to about50 mg/kg or more (usually up to about 100 mg/kg), generally dependingupon the pharmacokinetic of the two agents in the patient. These dosageranges generally produce effective blood level concentrations of activecompound in the patient.

For purposes of the present invention, a prophylactically or preventiveeffective amount of the compositions according to the present inventionfalls within the same concentration range as set forth above fortherapeutically effective amount and is usually the same as atherapeutically effective amount.

Administration of Compound 2 may range from continuous (intravenousdrip) to several oral or intranasal administrations per day (forexample, Q.I.D.) or transdermal administration and may include oral,topical, parenteral, intramuscular, intravenous, sub-cutaneous,transdermal (which may include a penetration enhancement agent), buccal,and suppository administration, among other routes of administration.Enteric coated oral tablets may also be used to enhance bioavailabilityof the compounds for an oral route of administration. The most effectivedosage form will depend upon the bioavailability/pharmacokinetic of theparticular agent chosen as well as the severity of disease in thepatient. Oral dosage forms are particularly preferred, because of easeof administration and prospective favorable patient compliance.

To prepare the pharmaceutical compositions according to the presentinvention, a therapeutically effective amount of Compound 2 according tothe present invention is often intimately admixed with apharmaceutically acceptable carrier according to conventionalpharmaceutical compounding techniques to produce a dose. A carrier maytake a wide variety of forms depending on the form of preparationdesired for administration, e.g., oral or parenteral. In preparingpharmaceutical compositions in oral dosage form, any of the usualpharmaceutical media may be used. Thus, for liquid oral preparationssuch as suspensions, elixirs, and solutions, suitable carriers andadditives including water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, and the like may be used. For solid oralpreparations such as powders, tablets, capsules, and for solidpreparations such as suppositories, suitable carriers and additivesincluding starches, sugar carriers, such as dextrose, manifold, lactose,and related carriers, diluents, granulating agents, lubricants, binders,disintegrating agents, and the like may be used. If desired, the tabletsor capsules may be enteric-coated or sustained release by standardtechniques. The use of these dosage forms may significantly enhance thebioavailability of the compounds in the patient.

For parenteral formulations, the carrier will usually comprise sterilewater or aqueous sodium chloride solution, though other ingredients,including those which aid dispersion, also may be included. Of course,where sterile water is to be used and maintained as sterile, thecompositions and carriers must also be sterilized. Injectablesuspensions may also be prepared, in which case appropriate liquidcarriers, suspending agents, and the like may be employed.

Liposomal suspensions (including liposomes targeted to viral antigens)may also be prepared by conventional methods to produce pharmaceuticallyacceptable carriers. This may be appropriate for the delivery of freenucleosides, acyl/alkyl nucleosides or phosphate ester pro-drug forms ofthe nucleoside compounds according to the present invention.

In typical embodiments according to the present invention, Compound 2and the compositions described are used to treat, prevent or delay a HCVinfection or a secondary disease state, condition or complication ofHCV.

VII. Combination and Alternation Therapy

It is well recognized that drug-resistant variants of viruses can emergeafter prolonged treatment with an antiviral agent. Drug resistancesometimes occurs by mutation of a gene that encodes for an enzyme usedin viral replication. The efficacy of a drug against an HCV infection,can be prolonged, augmented, or restored by administering the compoundin combination or alternation with another, and perhaps even two orthree other, antiviral compounds that induce a different mutation or actthrough a different pathway, from that of the principle drug.Alternatively, the pharmacokinetic, bio distribution, half-life, orother parameter of the drug can be altered by such combination therapy(which may include alternation therapy if considered concerted). Sincethe disclosed Compound 2 is an NS5B polymerase inhibitor, it may beuseful to administer the compound to a host in combination with, forexample a

-   -   (1) Protease inhibitor, such as an NS3/4A protease inhibitor;    -   (2) NS 5A inhibitor;    -   (3) Another NS5B polymerase inhibitor;    -   (4) NS5B non-substrate inhibitor;    -   (5) Interferon alfa-2a, which may be pegylated or otherwise        modified, and/or ribavirin;    -   (6) Non-substrate-based inhibitor;    -   (7) Helicase inhibitor;    -   (8) Antisense oligodeoxynucleotide (S-ODN);    -   (9) Aptamer;    -   (10) Nuclease-resistant ribozyme;    -   (11) iRNA, including microRNA and SiRNA;    -   (12) Antibody, partial antibody or domain antibody to the virus,        or    -   (13) Viral antigen or partial antigen that induces a host        antibody response.        Non limiting examples of anti-HCV agents that can be        administered in combination with Compound 2 of the invention,        alone or with multiple drugs from this lists, are

(i) protease inhibitors such as telaprevir (Incivek®), boceprevir(Victrelis™), simeprevir (Olysio™), paritaprevir (ABT-450), glecaprevir(ABT-493), ritonavir (Norvir), ACH-2684, AZD-7295, BMS-791325,danoprevir, Filibuvir, GS-9256, GS-9451, MK-5172, Setrobuvir,Sovaprevir, Tegobuvir, VX-135, VX-222, and, ALS-220;

(ii) NS5A inhibitor such as ACH-2928, ACH-3102, IDX-719, daclatasvir,ledispasvir, velpatasvir (Epclusa), elbasvir (MK-8742), grazoprevir(MK-5172), and Ombitasvir (ABT-267);

(iii) NS5B inhibitors such as AZD-7295, Clemizole, dasabuvir (Exviera),ITX-5061, PPI-461, PPI-688, sofosbuvir (Sovaldi®), MK-3682, andmericitabine;

(iv) NS5B inhibitors such as ABT-333, and MBX-700;

(v) Antibody such as GS-6624;

(vi) Combination drugs such as Harvoni (ledipasvir/sofosbuvir); ViekiraPak (ombitasvir/paritaprevir/ritonavir/dasabuvir); Viekirax(ombitasvir/paritaprevir/ritonavir); G/P (paritaprevir and glecaprevir);Technivie (ombitasvir/paritaprevir/ritonavir) and Epclusa(sofosbuvir/velpatasvir) and Zepatier (elbasvir and grazoprevir).

If Compound 2 is administered to treat advanced hepatitis C virusleading to liver cancer or cirrhosis, in one embodiment, the compoundcan be administered in combination or alternation with another drug thatis typically used to treat hepatocellular carcinoma (HCC), for example,as described by Andrew Zhu in “New Agents on the Horizon inHepatocellular Carcinoma” Therapeutic Advances in Medical Oncology, V5(1), January 2013, 41-50. Examples of suitable compounds forcombination therapy where the host has or is at risk of HCC includeanti-angiogenic agents, sunitinib, brivanib, linifanib, ramucirumab,bevacizumab, cediranib, pazopanib, TSU-68, lenvatinib, antibodiesagainst EGFR, mTor inhibitors, MEK inhibitors, and histone decetylaceinhibitors.

EXAMPLES General Methods

¹H, ¹⁹F and ³¹P NMR spectra were recorded on a 400 MHz Fourier transformBrucker spectrometer. Spectra were obtained DMSO-d₆ unless statedotherwise. The spin multiplicities are indicated by the symbols s(singlet), d (doublet), t (triplet), m (multiplet) and, br (broad).Coupling constants (J) are reported in Hz. The reactions were generallycarried out under a dry nitrogen atmosphere using Sigma-Aldrichanhydrous solvents. All common chemicals were purchased from commercialsources.

The following abbreviations are used in the Examples:

AUC: Area under the Curve

C₂₄: Concentration of the drug in plasma at 24 hours

C_(24,ss): Concentration at 24 hours after dosing at steady state

C_(max): Maximum concentration of the drug achieved in plasma

DCM: Dichloromethane

EtOAc: Ethyl acetate

EtOH: Ethanol

HPLC: High pressure liquid chromatography

NaOH: Sodium hydroxide

Na₂SO₄: Sodium sulphate (anhydrous)

MeCN: Acetonitrile

MeNH₂: Methylamine

MeOH: Methanol

Na₂SO₄: Sodium sulfate

NaHCO₃: Sodium bicarbonate

NH₄Cl: Ammonium chloride

NH₄OH: Ammonium hydroxide

PE: Petroleum ether

Ph₃P: Triphenylphosphine

RH: relative humidity

Silica gel (230 to 400 mesh, Sorbent)

t-BuMgCl: t-Butyl magnesium chloride

T_(max): Time at which C_(max) is achieved

THF: Tetrahydrofuran (THF), anhydrous

TP: Triphosphate

Example 1. Synthesis of Compound 1

Step 1: Synthesis of(2R,3R,4R,5R)-5-(2-Amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol(2-2)

A 50 L flask was charged with methanol (30 L) and stirred at 10±5° C.NH₂CH₃ (3.95 Kg) was slowly ventilated into the reactor at 10±5° C.Compound 2-1 (3.77 kg) was added in batches at 20±5° C. and stirred for1 hour to obtain a clear solution. The reaction was stirred for anadditional 6-8 hours, at which point HPLC indicated that theintermediate was less than 0.1% of the solution. The reactor was chargedwith solid NaOH (254 g), stirred for 30 minutes and concentrated at50±5° C. (vacuum degree: −0.095). The resulting residue was charged withEtOH (40 L) and re-slurried for 1 hour at 60° C. The mixture was thenfiltered through celite and the filter cake was re-slurried with EtOH(15 L) for 1 hour at 60° C. The filtrate was filtered once more,combined with the filtrate from the previous filtration, and thenconcentrated at 50±5° C. (vacuum degree: −0.095). A large amount ofsolid was precipitated. EtOAc (6 L) was added to the solid residue andthe mixture was concentrated at 50±5° C. (vacuum degree: −0.095). DCMwas then added to the residue and the mixture was re-slurried at refluxfor 1 hour, cooled to room temperature, filtered, and dried at 50±5° C.in a vacuum oven to afford compound 2-2 as an off-white solid (1.89 Kg,95.3%, purity of 99.2%).

Analytic Method for Compound 2-2: The purity of compound 2-2 (15 mg) wasobtained using an Agilent 1100 HPLC system with a Agilent Poroshell 120EC-C18 4.6*150 mm 4-Micron column with the following conditions: 1mL/min flow rate, read at 254 nm, 30° C. column temperature, 15 μLinjection volume, and a 31 minute run time. The sample was dissolved inacetonitrile—water (20:80) (v/v). The gradient method is shown below.

Time (min) A % (0.05 TFA in water) B % (Acetonitrile) 0 95 5 8 80 20 1350 50 23 5 95 26 5 95 26.1 95 5 31 95 5Step 2: Synthesis ofisopropyl((S)-(((2R,3R,4R,5R)-5-(2-Amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate(Compound 1)

Compound 2-2 and compound 2-3 (isopropyl((perfluorophenoxy)(phenoxy)phosphoryl)-L-alaninate) were dissolved inTHF (1 L) and stirred under nitrogen. The suspension was then cooled toa temperature below −5° C. and a 1.7 M solution of t-BuMgCl solution(384 mL) was slowly added over 1.5 hours while a temperature of 5-10° C.was maintained. A solution of NH₄Cl (2 L) and water (8 L) was added tothe suspension at room temperature followed by DCM. The mixture wasstirred for 5 minutes before a 5% aqueous solution of K₂CO₃ (10 L) wasadded and the mixture was stirred for 5 additional minutes beforefiltering through diatomite (500 g). The diatomite was washed with DCMand the filtrate was separated. The organic phase was washed with a 5%aqueous K₂CO₃ solution (10 L×2), brine (10 L×3), and dried over Na₂SO₄(500 g) for approximately 1 hour. Meanwhile, this entire process wasrepeated 7 times in parallel and the 8 batches were combined. Theorganic phases were filtered and concentrated at 45±5° C. (vacuum degreeof 0.09 Mpa). EtOAc was added and the mixture was stirred for 1 hour at60° C. and then at room temperature for 18 hours. The mixture was thenfiltered and washed with EtOAc (2 L) to afford crude Compound 1. Thecrude material was dissolved in DCM (12 L), heptane (18 L) was added at10-20° C., and the mixture was allowed to stir for 30 minutes at thistemperature. The mixture was filtered, washed with heptane (5 L), anddried at 50±5° C. to afford pure Compound 1 (1650 g, 60%).

Analytic Method for Compound 1: The purity of Compound 1 (25 mg) wasobtained using an Agilent 1100 HPLC system with a Waters XTerra Phenyl 5μm 4.6*250 mm column with the following conditions: 1 mL/min flow rate,read at 254 nm, 30° C. column temperature, 15 μL injection volume, and a25 minute run time. The sample was dissolved in acetonitrile—water(50:50) (v/v). The gradient method is shown below.

Time (min) A % (0.1% H₃PO₄ in water) B % (Acetonitrile) 0 90 10 20 20 8020.1 90 10 25 90 10

Example 2. Characterization of Amorphous and Crystalline Compound 1

Amorphous Compound 1 and crystalline Compound 1 were initially analyzedby XRPD, ¹HNMR, and HPLC. The XRPD patterns for both compounds are shownin FIG. 1A and the HPLC traces to determine purity are shown in FIGS. 1Band 2A, respectively. Table 1 is a list of peaks from the XRPD ofcrystalline Compound 1 and Table 2 is a list of relative retention times(RTT) from the HPLC traces. Amorphous Compound 1 was 98.61% pure andcrystalline Compound 1 was 99.11% pure. Both compounds were a whitesolid. FIG. 2B is the TGA and DSC graphs of crystalline Compound 1. Forcrystalline Compound 1, an endotherm was observed at 88.6° C. and therewas a 7.8% mass loss from 80-110° C.

A sample of Compound 1 was recrystallized from EtOAc/hexane and drawnwith ORTEP. The absolute structure of Compound 1 was confirmed by therecrystallization of a single crystal. FIG. 3 is the ORTEP drawing ofCompound 1. Crystal data and measurement data are shown in Table 3. Theabsolute stereochemistry of Compound 1 based on the X-raycrystallography is shown below:

DSC data were collected on a TA Instruments Q2000 equipped with a 50position auto-sampler. The calibration for thermal capacity was carriedout using sapphire and the calibration for energy and temperature wascarried out using certified indium. Typically approximately 3 mg of eachsample, in a pin-holed aluminum pan, was heated at 10° C./min from 25°C. to 200° C. A purge of dry nitrogen at 50 ml/min was maintained overthe sample. The instrument control software was Advantage for Q Seriesv2.8.0.394 and Thermal Advantage v5.5.3 and the data were analyzed usingUniversal Analysis v4.5A.

TGA data were collected on a TA Instruments Q500 TGA, equipped with a 16position auto-sampler. The instrument was temperature calibrated usingcertified Alumel and Nickel. Typically 5-10 mg of each sample was loadedonto a pre-tared aluminum DSC pan and heated at 10° C./min from ambienttemperature to 350° C. A nitrogen purge at 60 ml/min was maintained overthe sample. The instrument control software was Advantage for Q Seriesv2.5.0.256 and Thermal Advantage v5.5.3 and the data were analyzed usingUniversal Analysis v4.5.

Amorphous Compound 1 (1-1):

¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.01-1.15 (m, 9 H), 1.21 (d, J=7.20 Hz,3 H), 2.75-3.08 (m, 3 H), 3.71-3.87 (m, 1 H), 4.02-4.13 (m, 1 H),4.22-4.53 (m, 3 H), 4.81 (s, 1 H), 5.69-5.86 (m, 1 H), 6.04 (br d,J=19.33 Hz, 4 H), 7.1 -7.27 (m, 3 H), 7.27-7.44 (m, 3 H), 7.81 (s, 1 H)

Crystalline Compound 1 (1-2):

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.97-1.16 (m, 16 H), 1.21 (d, J=7.07 Hz,3 H), 2.87 (br s, 3 H), 3.08 (s, 2 H), 3.79 (br d, J=7.07 Hz, 1 H), 4.08(br d, J=7.58 Hz, 1 H), 4.17-4.55 (m, 3 H), 4.81 (quin, J=6.25 Hz, 1 H),5.78 (br s, 1 H), 5.91-6.15 (m, 4 H), 7.10-7.26 (m, 3 H), 7.26-7.44 (m,3 H), 7.81 (s, 1 H)

TABLE 1 Peak list for crystalline Compound 1 Angle/°2θ d spacing/ÅIntensity/Counts Intensity/% 6.03 14.64 1005 39.0 7.36 12.00 315 12.27.94 11.13 1724 66.9 9.34 9.47 2500 97.0 9.51 9.29 860 33.4 9.77 9.051591 61.8 11.08 7.98 2576 100.0 12.02 7.36 171 6.6 12.95 6.83 319 12.413.98 6.33 241 9.4 14.30 6.19 550 21.4 14.69 6.03 328 12.7 15.20 5.822176 84.5 15.94 5.56 1446 56.1 16.75 5.29 1009 39.2 17.29 5.13 700 27.217.72 5.00 1213 47.1 18.11 4.89 1565 60.8 18.46 4.80 302 11.7 18.89 4.69385 14.9 19.63 4.52 636 24.7 20.37 4.36 1214 47.1 20.74 4.28 1198 46.521.24 4.18 640 24.8 22.31 3.98 961 37.3 22.88 3.88 806 31.3 23.43 3.79355 13.8 24.08 3.69 573 22.2 24.49 3.63 159 6.2 25.00 3.56 351 13.625.36 3.51 293 11.4 26.09 3.41 235 9.1 26.26 3.39 301 11.7 26.83 3.32696 27.0 27.35 3.26 436 16.9 27.46 3.25 363 14.1 28.07 3.18 200 7.828.30 3.15 195 7.6 28.82 3.10 599 23.3 29.85 2.99 217 8.4 30.26 2.95 1867.2 30.75 2.91 333 12.9 31.12 2.87 149 5.8 31.85 2.81 238 9.2 33.28 2.69261 10.1 34.77 2.58 171 6.6 35.18 2.55 175 6.8 36.83 2.44 327 12.7 37.412.40 172 6.7

TABLE 2 Relative Retention Times from HPLC chromatographs of AmorphousCompound 1 and Crystalline Compound 1 Amorphous Compound 1 CrystallineCompound 1 RRT Area % RRT Area % 0.48 0.15 0.48 0.17 0.51 0.04 0.48 0.170.48 0.15 0.94 0.12 0.51 0.04 1.00 99.11 0.94 0.13 1.04 0.22 0.98 0.211.37 0.07 1.00 98.61 1.04 0.29 1.37 0.31

TABLE 3 Crystal and Data Measurement of Compound 1 Bond Precision C-C =0.0297 A, Wavelength = 1.54184 Cell a = 10.1884(3) b = 28.6482(9) c =12.9497(5) alpha = 90 beta = 113.184(4) gamma = 90 Temperature 150 KCalculated Reported Volume 3474.5 (2) 3474.5 (2) Space Group P21 P 1 211 Hall Group P 2yb P 2yb Moiety Formula C24 H34 F N7 O7 P 2(C24 H34 F N7O7 P) Sum Formula C24 H34 F N7 O7 P C48 H68 F2 N14 O14 P2 Mr 582.551165.10 Dx, g cm⁻¹ 1.114 1.114 Z 4 2 Mu (mm⁻¹) 1.139 1.139 F000 1228.01228.0 F000′ 1233.21 h, k, l_(max) 12, 34, 15 12, 34, 15 N_(ref) 12742[6510] 8259 T_(min), T_(max) 0.790, 0.815 0.808, 1.000 T_(min′) 0.716Correction Method # Reported T Limits: T_(min) = 0.808 T_(max) = 1.00AbsCorr MULTI-SCAN Data completeness 1.27/0.65 Theta (max) 68.244 R(reflections) 0.2091 (7995) wR2 (reflections) 0.5338 (8259) S 2.875 Npar716

This initial characterization was followed by storage at 25° C./60%relative humidity (RH) for 14 days with analysis by HPLC and XRPD after7 and 14 days. FIG. 4A is the XRPD after 14 days at 25° C./60% (RH).Amorphous Compound 1 (sample 1-1) remained poorly crystalline, whereascrystalline Compound 1 (sample 1-2) retained its crystallinity, but bothcompounds were stable after 14 days at 25° C./60% (RH).

Example 3. Formation of Oxalate Salt Compound 4

Initially, the oxalate salt of Compound 1, Compound 4, was formed bymixing the oxalic salt with solvent (5 vol, 100 μL) and allowing anysolution to evaporate at room temperature. Any suspension was matured(room temperature—50° C.) for 3 hours and crystallinity was accessed.

Table 4 shows the different solvents used in the production of Compound4. All solvents except for two (cyclohexane and n-heptane) affordedcrystalline products. Despite the high crystallinity and solubility ofCompound 4, oxalate salts are not acceptable for clinical developmentdue to the potential formation of kidney stones and other salts ofcompound 1 were explored.

TABLE 4 Formation of Oxalate Compound 4 Observation post acidObservation after addition at room maturation/ Solvent temperatureevaporation EtOH Solution OXA - Form 1 IPA Solution OXA - Form 1 AcetoneSolution OXA - Form 1 MEK Solution OXA - Form 1 EtOAc Suspension OXA -Form 1 iPrOAc Suspension OXA - Form 1 THF Solution OXA - Form 1 TolueneSolution OXA - Form 1 MeCN Solution OXA - Form 1 IPA: 10% water SolutionOXA - Form 1 TBME Suspension OXA - Form 1 Cyclohexane SuspensionAmorphous n-Heptane Suspension Amorphous

Example 4. Salt Compounds of Amorphous Compound 1

Since the oxalate salt compound 4 (Example 3) could not be carriedforward in clinical trials due to its potential to form kidney stones,amorphous salts of Compound 1 were formed with the counter ions listedin Table 5. Compound 1 was dissolved in t-butanol (20 vol, 6 ml) and thesolution was treated with the acid counter-ions (1 equivalent for eachsample except sample 1-9 which had 0.5 equivalent of sulfate). Thesamples were then frozen with the solvent removed by lyophilization. Theresidual solid in samples 1-4, 1-5, 1-6, 1-7, 1-8, and 1-9 was initiallyanalyzed by XRPD and HPLC.

TABLE 5 Amorphous salt formation details Stock Sample Sample solution IDdetails details Observation NMR 1-4 HCl (1:1) THF 1M White solid 3 fewerprotons ~0.3 eq t-BuOH 1-5 Sulfuric THF 1M White solid 3 fewer protons(1:1) ~0.3 eq t-BuOH 1-6 Fumaric MeOH:THF Glassy solid 1.05 eq fumaricacid (1:1) (1:1) 0.5M 0.84 eq t-BuOH 1-7 Benzoic THF White solid 1.0 eqbenzoic acid (1:1) 1M 0.34 eq t-BuOH 1-8 Succinic MeOH Sticky white ~1.1eq succinic acid (1:1) 1M solid 0.37 eq t-BuOH 1-9 Sulfuric THF Whitesolid 3 fewer protons (0.5:1 1M ~0.3 eq t-BuOH acid:API)¹HNMR spectrum were taken for all samples.

Sample 1-4, HCl (1:1) Salt:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.93-1.39 (m, 16 H), 2.97 (br s, 2 H),3.70-3.88 (m, 1 H), 4.10 (br s, 1 H), 4.18-4.49 (m, 3 H), 4.70-4.88 (m,1 H), 5.71-5.94 (m, 1 H), 6.07 (br d, J=19.07 Hz, 2 H), 7.14-7.27 (m, 3H), 7.29-7.44 (m, 2 H), 7.83-8.19 (m, 1H)

Sample 1-5, Sulfuric (1:1) Salt:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.97-1.38 (m, 15 H), 2.96 (br s, 2 H),4.06-4.18 (m, 1 H), 4.19-4.49 (m, 3 H), 4.66-4.91 (m, 1 H), 5.70-5.95(m, 1 H), 5.96-6.16 (m, 2 H), 7.10-7.27 (m, 3 H), 7.30-7.43 (m, 2 H),7.88-8.19 (m, 1H)

Sample 1-6, Fumaric (1:1) Salt:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.95-1.31 (m, 21 H), 2.87 (br s, 3 H),3.79 (br d, J=7.20 Hz, 1 H), 4.01-4.13 (m, 1 H), 4.16-4.23 (m, 1 H),4.16-4.24 (m, 1 H), 4.20 (s, 1 H), 4.18-4.23 (m, 1 H), 4.24-4.52 (m, 1H), 4.24-4.52 (m, 1 H), 4.24-4.49 (m, 1 H), 4.72-4.88 (m, 1 H),5.68-5.86 (m, 1 H), 6.04 (br d, J=19.33 Hz, 4 H), 6.63 (s, 1 H),6.61-6.66 (m, 1 H), 7.12-7.27 (m, 3 H), 7.27-7.45 (m, 3 H), 7.81 (s, 1H), 13.16 (br s, 2 H)

Sample 1-7, Benzoic (1:1) Salt:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.96-1.30 (m, 15 H), 2.87 (br s, 3 H),3.79 (br d, J=7.07 Hz, 1 H), 4.07 (br s, 1 H), 4.20 (s, 1 H), 4.25-4.52(m, 3 H), 4.81 (s, 1 H), 5.71-5.85 (m, 1 H), 6.04 (br d, J=19.33 Hz, 4H), 7.08-7.27 (m, 3 H), 7.27-7.43 (m, 3 H), 7.45-7.57 (m, 2 H), 7.63 (s,1 H), 7.81 (s, 1 H), 7.95 (dd, J=8.27, 1.33 Hz, 2 H), 12.98 (br s, 1 H)

Sample 1-8, Succinic (1:1) Salt:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.98-1.28 (m, 15 H), 2.42 (s, 5 H), 2.87(br s, 3 H), 3.57-3.62 (m, 1 H), 3.70-3.86 (m, 1 H), 4.02-4.14 (m, 1 H),4.20 (s, 1 H), 4.24-4.51 (m, 3 H), 4.70-4.88 (m, 1 H), 5.69-5.86 (m, 1H), 6.04 (br d, J=19.33 Hz, 4 H), 7.12-7.27 (m, 3 H), 7.27-7.44 (m, 3H), 7.81 (s, 1 H), 11.95-12.58 (m, 2 H)

Sample 1-9, Sulfuric (0.5:1) Salt:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.02-1.31 (m, 15 H), 2.94 (br s, 3 H),3.79 (br d, J=7.20 Hz, 2 H), 4.09 (br s, 1 H), 4.22-4.48 (m, 3 H),4.72-4.90 (m, 1 H), 5.71-5.92 (m, 1 H), 6.07 (br d, J=19.07 Hz, 2 H),7.12-7.28 (m, 3 H), 7.31-7.44 (m, 2 H), 7.75-8.19 (m, 1 H).

The samples were then subjected to storage at 25° C./60% relativehumidity (RH) for 14 days with analysis by HPLC and XRPD after 7 (FIG.4B) and 14 days (FIG. 5A). All prepared salts remained amorphous and theobservations are shown in Table 6. The mono sulfate (sample 1-5) andsuccinate salts (sample 1-8) were found to be physically unstable anddeliquesced or became a gum during the course of the study. Both thefumarate (sample 1-6) and benzoate salts (sample 1-7) were found to beglassy solids. The HCl salt (sample 1-4) was found to retain itsphysical appearance. Surprisingly, the hemi-sulfate salt (sample 1-9)also retained its physical appearance as a white solid in contrast tomono-sulfate compound (sample 1-5), which was a sticky gum. Results areshown in Table 6. The mono HCl salt (sample 1-4) and the hemi-sulfatesalt (sample 1-9) were found to be physically and chemically stableafter 2 weeks storage at 25° C./60% relative humidity (RH). Althoughboth salts were stable over the two weeks, the hemi-sulfate salt wassuperior to the HCl salt because the HCl salt was hygroscopic, renderingit less useful compared to the hemi-sulfate salt for long-term storageor use.

TABLE 6 Stability of samples after 7 and 14 days at 25° C./60% RH Timeexposed to 25° C./60% RH (days) 0 7 14 Sample ID HPLC Observation HPLCObservation HPLC Observation 1-1 98.6 White solid 98.7 White solid 98.5White solid 1-2 99.1 White solid 99.2 White solid 99.0 White solid 1-399.7 White solid 99.6 White solid 99.4 White solid 1-4 98.7 White solid98.8 White solid 98.6 White solid 1-5 98.4 White solid 55.7 Sticky white— Sticky gum solid 1-6 98.7 Glassy solid 98.6 Clear glassy 98.4 Whiteglassy solid solid 1-7 98.8 White solid 98.8 Clear glassy 98.7 Clearglassy solid solid 1-8 98.7 Sticky white — Deliquesced/ — Deliquescedsolid sticky oil 1-9 98.7 White solid 98.1 White solid 96.4 White solid

Example 5. Characterization of Amorphous Compound 2

Amorphous Compound 2 was initially analyzed by XRPD, ¹HNMR, DSC, TGA,and HPLC. The XRPD pattern for amorphous Compound 2 overlaid withamorphous Compound 1 and crystalline Compound 1 is shown in FIG. 1A andthe XRPD pattern of amorphous Compound 2 alone is shown in FIG. 5B.Table 7 is a peak list from the XRPD pattern shown in FIG. 5B. The HPLCtrace to determine purity is shown in FIG. 6A. Table 8 is a list ofrelative retention times (RTT) from the HPLC trace shown in FIG. 6A.Amorphous Compound 2 was 99.68% pure. FIG. 6B is a TGA and DSC graph ofamorphous Compound 2. Experimental details for the TGA and DSCexperiments are given in Example 2.

TABLE 7 Peak list for Amorphous Compound 2 Angle/°2θ d spacing/ÅIntensity/Counts Intensity/% 4.20 21.03 486 81.8 4.67 18.91 482 81.05.16 17.10 595 100.0 9.13 9.68 547 92.0

TABLE 8 HPLC chromatogram of Amorphous Compound 2 Amorphous Compound 2RRT Area % 0.48 0.02 0.48 0.02 0.67 0.01 0.94 0.13 1.00 99.68 1.04 0.06

Amorphous Compound 2:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.93-1.29 (m, 13 H), 2.94 (br s, 3 H),3.79 (td, J=10.04, 7.07 Hz, 2 H), 4.05-4.19 (m, 1 H), 4.19-4.50 (m, 3H), 4.81 (quin, J=6.25 Hz, 1 H), 5.71-5.94 (m, 1 H), 5.97-6.16 (m, 2 H),7.14-7.28 (m, 3 H), 7.31-7.44 (m, 2 H), 7.82-8.09 (m, 1H)

Example 6. Crystallization of Amorphous Compound 2

Since the hemi-sulfate salt was found to remain as a solid after the 14day stability study as shown in Table 6, preliminary tests studyingcrystallization conditions using 11 different solvents was conducted.Amorphous Compound 2 was suspended in 5 volumes of solvent at 25° C.(sample 2-1, 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, and 2-11). Tothose samples that were not free flowing (2-1, 2-2, 2-3, 2-4, 2-5, 2-6,2-7, 2-8, and 2-10), an additional 5 volumes of solvent was added. Thesamples were then matured at 25-50° C. (1° C./min between temperaturesand 4 hour at each temperature) for 6 days except for sample 2-1, whichwas observed to be a clear solution after 1 day and was allowed toevaporate under ambient conditions. The results are shown in Table 9.Crystalline patterns resulted from crystallization with isobutanol(sample 2-1), acetone (sample 2-2), EtOAc (sample 2-6), and iPrOAc(sample 2-7). Two poorly crystalline samples were also identified fromcrystallization with MEK (sample 2-4) and MIBK (sample 2-5). The XRPDpatterns are shown in FIG. 7A.

TABLE 9 Crystallization Conditions of Compound 2 Sam- ObservationObservation Observation ple after 5 after 10 after 1 day ID Solventvolumes volumes maturation XRPD 2-1 IPA Solid - not Free flowingSolution, Gum free flowing suspension evaporated at RT yielding a gum2-2 Iso- Solid - not Free flowing Suspension Crystalline - butanol freeflowing suspension Pattern 2 2-3 Acetone Solid - not Free flowingSuspension Crystalline - free flowing suspension Pattern 3 2-4 MEKSolid - not Free flowing Suspension Poorly free flowing suspensioncrystalline - Pattern 4 2-5 MIBK Solid - not Free flowing SuspensionPoorly free flowing suspension crystalline - Pattern 4 2-6 EtOAc Solid -not Free flowing Suspension Crystalline - free flowing suspensionPattern 1 2-7 iPrOAc Solid - not Free flowing Suspension Crystalline -free flowing suspension Pattern 1 2-8 THF Solid - not Free flowingSuspension Poorly free flowing suspension crystalline 2-9 TBME Freeflowing — Suspension Amorphous suspension 2-10 Toluene Solid - not Freeflowing Suspension Amorphous free flowing suspension 2-11 Heptane Freeflowing — Suspension Amorphous suspension

The seven samples (Samples 2-2, 2-3, 2-4, 2-5, 2-6, 2-7 and 2-8) wereanalyzed by DSC, TGA, ¹H-NMR and IC (Table 10, FIG. 8A, FIG. 8B, FIG.9A, FIG. 9B, FIG. 10A, FIG. 10B, FIG. 11A, and FIG. 11B) as well as byXRPD following 6 days storage at 25° C./60% relative humidity (RH) (allsamples remained crystalline/poorly crystalline following stability).All samples retained roughly half an equivalent of sulfate, butcontained a relatively large amount of residual solvent. An overlay ofthe X-ray diffractograms of amorphous samples 2-9, 2-10, and 2-11 isshown in FIG. 7B.

TABLE 10 Characterization of crystalline Compound 2 samples IC Sample(corrected ID Solvent DSC TGA ¹HNMR for TGA) 2-2 Isobutanol Endo 113.8°C. 8.3% 1.1 eq 0.45 eq ambient- isobutanol 140° C. 2-3 Acetone Endo30-95° C. 7.6% 0.5 eq acetone 0.46 eq Endo 100-145° C. ambient- 140° C.2-4 MEK Broad complex 8.5% 0.8 eq MEK 0.45 eq endo 30-115° C. ambient-Endo 115-145° C. 140° C. 2-5 MIBK Broad endo 30-105° C. 5.2% 0.2 eq MIBK0.46 eq Endo 114.7° C. ambient- 110° C. 2-6 EtOAc Sharp endo 113.6° C.2.0% 0.9 eq EtOAc 0.46 eq ambient- 100° C. 2-7 iPrOAc Endo 30-90° C.1.6% 0.8 eq iPrOAc 0.45 eq ambient- 90° C. 2-8 THF Endo 30-100° C. 4.2%0.7 eq THF 0.45 eq Sharper endo 115.6° C. ambient- 130° C.¹HNMR spectrum were taken for all samples and listed below.

Sample 2-2:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.83 (d, J=6.69 Hz, 7 H), 0.99-1.26 (m,14 H), 1.61 (dt, J=13.26, 6.63 Hz, 1 H), 3.73-3.87 (m, 2 H), 4.03-4.18(m, 1 H), 4.18-4.51 (m, 4 H), 4.66-4.92 (m, 1 H), 4.70-4.90 (m, 1 H),4.72-4.88 (m, 1 H), 5.81 (br s, 1 H), 5.93-6.11 (m, 2 H), 7.10-7.26 (m,3 H), 7.14-7.26 (m, 1 H), 7.30-7.41 (m, 2 H), 7.94 (br s, 1 H)

Sample 2-3:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.00-1.26 (m, 13 H), 2.09 (s, 3 H),3.74-3.87 (m, 2 H), 4.10 (br d, J=7.70 Hz, 1 H), 4.22-4.50 (m, 3 H),4.81 (quin, J=6.28 Hz, 1 H), 5.71-5.90 (m, 1 H), 5.96-6.15 (m, 2 H),7.12-7.26 (m, 3 H), 7.31-7.41 (m, 2 H), 7.79-8.07 (m, 1 H)

Sample 2-4:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.91 (t, J=7.33 Hz, 3 H), 1.01-1.28 (m,13 H), 2.08 (s, 2 H), 3.72-3.89 (m, 2 H), 4.10 (br d, J=8.08 Hz, 1 H),4.23-4.47 (m, 3 H), 4.81 (quin, J=6.25 Hz, 1 H), 5.69-5.89 (m, 1 H),5.94-6.13 (m, 2 H), 7.14-7.25 (m, 3 H), 7.32-7.41 (m, 2 H), 7.79-8.11(m, 1 H)

Sample 2-5:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.86 (d, J=6.69 Hz, 1 H), 0.98-1.33 (m,13 H), 2.02-2.09 (m, 1 H), 4.03-4.17 (m, 1 H), 4.22-4.50 (m, 3 H), 4.81(quin, J=6.25 Hz, 1 H), 5.81 (br s, 1 H), 5.93-6.15 (m, 2 H), 7.11-7.27(m, 3 H), 7.31-7.41 (m, 2 H), 7.77-8.21 (m, 1 H)

Sample 2-6:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.98-1.28 (m, 15 H), 2.00 (s, 3 H),3.99-4.14 (m, 3 H), 4.21-4.49 (m, 3 H), 4.81 (quin, J=6.22 Hz, 1 H),5.82 (br s, 1 H), 5.93-6.14 (m, 2 H), 7.11-7.26 (m, 3 H), 7.29-7.42 (m,2 H), 7.79-8.17 (m, 1H)

Sample 2-7:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.92-1.28 (m, 17 H), 1.97 (s, 2 H),4.04-4.16 (m, 1 H), 4.20-4.51 (m, 3 H), 4.71-4.93 (m, 2 H), 5.82 (br s,1 H), 5.95-6.14 (m, 2 H), 7.11-7.28 (m, 3 H), 7.31-7.43 (m, 2 H),7.75-8.21 (m, 1 H)

Sample 2-8:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.81-1.11 (m, 13 H), 1.19 (s, 1 H),1.53-1.66 (m, 1 H), 3.87-4.01 (m, 1 H), 4.06-4.32 (m, 3 H), 4.64 (quin,J=6.25 Hz, 1 H), 5.55-5.75 (m, 1 H), 5.77-5.97 (m, 2 H), 6.94-7.10 (m, 3H), 7.13-7.26 (m, 2 H), 7.66-7.96 (m, 1 H)

Example 7. Failure to Crystallize Amorphous Malonate Salt (Compound 4)

As shown in Example 3, a crystalline oxalate salt was identified whendetermining appropriate salts for Compound 1, but oxalate salt Compound4 could not be carried forward in clinical trials due to its potentialfor causing kidney stones. Therefore, crystallization of the chemicallyrelated malonate salt (Compound 5) was attempted using the same 11solvents as for the hemi-sulfate salt. Compound 1 (12×50 mg, samples3-1, 3-2, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, and 3-12) wasdissolved in t-butanol (20 vol) and the solutions were then treated with1 equivalence of a malonic acid stock solution (1 M in THF). The sampleswere then frozen with the solvent removed by lyophilisation. To samples3-1, 3-2, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, and 3-11, relevantsolvent (5 volumes) was added at room temperature. Any resultingsolutions were allowed to evaporate under ambient conditions, while gumsor solids were matured at 25-50° C. (1° C./min between temperatures and4 hour at each temperature) for 5 days. The solids were analyzed by XRPD(FIG. 12B), but all samples were found to either form a gum or wereamorphous (FIG. 12B). Results are shown in Table 11. The one solid(amorphous) sample (3-12) was analyzed by ¹H-NMR and HPLC, and was foundto contain around 1 equivalence of malonic acid (peaks overlap) as wellas 0.6 eq. t-BuOH. The compound was 99.2% pure (FIG. 13A). FIG. 12A isan XRDP of sample 3-12 and FIG. 13A is the HPLC chromatograph of sample3-12.

Sample 3-12:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.81-1.11 (m, 13 H), 1.19 (s, 1 H),1.53-1.66 (m, 1 H), 3.87-4.01 (m, 1 H), 4.06-4.32 (m, 3 H), 4.64 (quin,J=6.25 Hz, 1 H), 5.55-5.75 (m, 1 H), 5.77-5.97 (m, 2 H), 6.94-7.10 (m, 3H), 7.13-7.26 (m, 2 H), 7.66-7.96 (m, 1 H)

TABLE 11 Crystallization Conditions of Amorphous Malonate Salt Compound4 Observation after 5 Sample Observation days maturation/ ID Solventafter 5 volumes evaporation XRPD 3-1 IPA Clear solution* Clear gum — 3-2Isobutanol Clear solution* Clear gum — 3-3 Acetone Clear solution* Cleargum — 3-4 MEK Clear solution* Clear gum — 3-5 MIBK Solution & Clear gum— some gum 3-6 EtOAc Clear solution* Clear gum & Amorphous crystal-likeappearance 3-7 iPrOAc Gum Clear gum — 3-8 THF Clear solution* Clear gum— 3-9 TBME Thick Clear gum — suspension 3-10 Toluene White gum/ Whitegum Amorphous solid 3-11 Heptane White solid White gum Amorphous(static) 3-12 — (White solid - (Sticky white solid - Amorphous nosolvent) ambient conditions) *Evaporated at room temperature

Example 8. Failure of Adequate Salt Formation using Liquid AssistedGrinding (LAG)

A liquid assisted grinding (LAG) study to determine appropriate saltsother than hemi-sulfate was performed using the 14 acidic counter ionsin Table 12.

TABLE 12 Counter-ion stock solutions used in LAG CrystallizationCounter-ion Solvent (1M) Pamoic DMSO Malonic THF D-Glucuronic WaterDL-Mandelic THF D-Gluconic THF Glycolic THF L-Lactic THF Oleic THFL-Ascorbic Water Adipic THF (heat) Caproic THF Stearic THF Palmitic THFMethane sulfonic THF

Compound 1 (30 mg) was placed in HPLC vials with two 3 mm ball bearings.The materials were wetted with solvent (15 μl ethanol, sample 4-1, 4-2,4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, and 4-14) and1 equivalence of the acid counter-ion was added. The samples were thenground for 2 hours at 650 rpm using a Fritsch milling system with anAutomaxion adapter. Most of the samples after grinding were found to beclear gums and were not analyzed further (Table 13). Those that wereobserved to contain solid were analyzed by XRPD and, in all cases, thepatterns obtained were found to match those of the crystalline acidcounter ion with no additional peaks (FIG. 13B).

TABLE 13 Observations and XRPD Results from LAG of Compounds 1 SampleObservation ID Acid after grinding XRPD 4-1 Pamoic Yellow gum/solidPamoic acid & amorphous halo 4-2 Malonic Clear gum — 4-3 D-GlucuronicWhite gum/solid D-Glucuronic acid & amorphous halo 4-4 DL-Mandelic Cleargum — 4-5 D-Gluconic Clear gum — 4-6 Glycolic Clear gum 4-7 L-LacticClear gum — 4-8 Oleic Clear gum — 4-9 L-Ascorbic White gum/solidL-Ascorbic acid & amorphous halo 4-10 Adipic Clear gum — 4-11 CaproicClear gum — 4-12 Stearic White gum/solid Stearic acid & amorphous halo4-13 Palmitic White gum/solid Palmitic acid & amorphous halo 4-4Methanesulfonic Clear gum —

Example 9. Failure to Obtain Adequate Salt Formation using Methyl EthylKetone (MEK)

Methyl ethyl ketone (MEK) was next utilized as a solvent to studyappropriate salts other than the hemi-sulfate salt. Using the 14 acidiccounter ions in Table 12, the study was performed by dissolving Compound1 (50 mg) in MEK (20 vol) at room temperature. The solutions weretreated with 1 equivalence of the selected counter-ions (Table 12). Thesamples were then cooled down to 5° C. at 0.1° C./min and stirred atthis temperature overnight. All samples were allowed to evaporate underambient conditions and any solids observed were analyzed by XRPD. Thisevaporation mainly produced gums, with the exception of the samples withsteric acid (sample 4-12) and palmitic acid (sample 5-13), whichafforded glassy solvents. These solids were amorphous by XRPD, but nocrystalline forms of the salt were obtained. Results are shown in Table14. (FIG. 13A).

TABLE 14 Results from dissolving Compound 1 in MEK (20 volumes)Observation Observation Sample Solvent upon acid Observation upon IDAcid for acid at 1M addition upon cooling evaporation 5-1 Pamoic DMSOYellow Yellow Yellow gum solution solution 5-2 Malonic THF SolutionSolution Clear gum 5-3 D-Glucuronic Water Solution Solution Clear gum5-4 DL-Mandelic THF Solution Solution Clear gum 5-5 D-Gluconic THF WhiteTurbid Clear gum precipitate solution 5-6 Glycolic THF Solution SolutionClear gum 5-7 L-Lactic THF Solution Solution Clear gum 5-8 Oleic THFSolution Solution Clear gum 5-9 L-Ascorbic Water Solution SolutionYellow gum 5-10 Adipic THF Solution Solution Clear gum (heat) 5-11Caproic THF Solution Solution Clear gum 5-12 Stearic THF Solution TurbidClear glassy solution solid* 5-13 Palmitic THF Solution Solution Clearglassy solid* 5-14 Methanesulfonic THF Solution Solution Clear gum Stocksolution prepared prior to acid addition *Samples were analyzed by XRPDand gave amorphous patterns plus peaks from the acid counter ion

Since all samples were amorphous, all samples were redissolved in MEK (5vol) and cyclohexane was added (20 vol antisolvent) at room temperaturefollowed by 1 hour of stirring at 25° C. The samples were then maturedbetween 50-5° C. (1° C./min between temperatures, 4 hours at eachtemperature) for 2 days before the cycle was changed to 50-25° C. for afurther 4 days. The samples were observed by eye following maturation.Results are shown in Table 15. Following the maturation, all samplesexcept 5-1 (with pamoic acid) were found to be gums. Sample 5-1, ayellow solid, was analyzed by XRPD, and the pattern was found to matchthe known form of pamoic acid (FIG. 14B), and therefore no crystallineforms of the salt were obtained.

TABLE 15 Results from redissolving Compound 1 in MEK (5 volumes) andantisolvent Observation Observation Sample Immediate Observation afterafter ID Observation after 10 minutes 60 minutes Maturation 5-1Precipitate Gum Gum Yellow suspension** 5-2 Precipitate Gum Gum Gum 5-3Precipitate/gum Gum Gum Gum 5-4 Precipitate Gum Gum Gum 5-5Precipitate/gum Gum Gum Gum 5-6 Precipitate Gum Gum Gum 5-7 PrecipitateGum Gum Gum 5-8 Precipitate Light suspension Gum Gum 5-9 Precipitate GumGum Gum 5-10 Precipitate Gum Gum Gum 5-11 Precipitate Light suspensionGum Gum 5-12 Precipitate Light suspension Gum Gum 5-13 Precipitate Lightsuspension Gum Gum 5-14 Precipitate Gum Gum Gum **Sample analyzed byXRPD with pattern matching known form of pamoic acid (no additionalpeaks

Example 10. Failure to Obtain Adequate Salt Formation using EthylAcetate

Ethyl acetate was next utilized to study appropriate salts other thanhemi-sulfate salt. Utilizing the 14 acidic counter ions in Table 12, thestudy was performed by dissolving Compound 1 (50 mg) in ethyl acetate(20 vol) at 50° C. The solutions were treated with 1 equivalent of theselected counter-ions (Table 12). The samples were then cooled down to5° C. at 0.1° C./min and stirred at this temperature for 4 days. Thesolutions were allowed to evaporate under ambient conditions while anysolids were analyzed by XRPD. The results from the crystallizationsusing ethyl acetate are in Table 16. In contrast to Example 8 where MEKwas the solvent, the majority of samples were observed to be suspensionsfollowing cooling of the acid:compound mixture (those that weresolutions were allowed to evaporate under ambient conditions). However,the XRPD diffractograms were generally found to match crystallineCompound 1. Samples 6-2, 6-4, and 6-5 have some slight differences (FIG.14A and FIG. 15A). No crystalline forms of the salt were obtained.

TABLE 16 Results from dissolving Compound 1 in EtOAc (20 volumes)Observation Observation Observation Solvent for upon acid upon uponSample ID Acid acid at 1M addition Cooling XRPD Evaporation 6-1 PamoicDMSO Yellow Yellow — Gum solution solution* 6-2 Malonic THF SolutionWhite Slight — suspension differences to freebase 6-3 D-Glucuronic WaterSolution Solution* — Gum 6-4 DL-Mandelic THF Solution White Slight —suspension differences to freebase 6-5 D-Gluconic THF White PossibleSlight — precipitate white gum differences to freebase 6-6 Glycolic THFSolution White Freebase — suspension 6-7 L-Lactic THF Solution WhiteFreebase — suspension 6-8 Oleic THF Solution White Freebase — suspension6-9 L-Ascorbic Water Solution Solution* — White solid on side/ yellowgum - amorphous 6-10 Adipic THF Solution White Freebase — (heat)suspension 6-11 Caproic THF Solution White Freebase — suspension 6-12Stearic THF Solution White Freebase — suspension 6-13 Palmitic THFSolution White Freebase — suspension 6-14 Methanesulfonic THF WhiteSolution/ — Clear gum precipitate clear gum*

Example 11. Chemical Purity Determination by HPLC

Purity analysis in Example 2 and Example 4 was performed on an AgilentHP1100 series system equipped with a diode array detector and usingChemStation software vB.04.03 using the method shown in Table 17.

TABLE 17 HPLC method for chemical purity determinations Parameter ValueType of method Reverse phase with gradient elution Sample Preparation0.5 mg/ml in acetonitrile:water 1:1 Column Supelco Ascentis Express C18,100 × 4.6 mm, 2.7 μm Column Temperature (° C.) 25  Injection (□l) 5Wavelength, Bandwidth (nm) 255, 90 Flow Rate (ml/min) 2 Phase A 0.1% TFAin water Phase B 0.085% TFA in acetonitrile Time (min) % Phase A % PhaseB Timetable 0 95 5 6 5 95 6.2 95 5 8 95 5

Example 12. X-Ray Powder Diffraction (XRPD) Techniques

The XRPD patterns in Examples 2, 3, 4, 5, 6, 7, 8, and 9 were collectedon a PANalytical Empyrean diffractometer using Cu K□ radiation (45 kV,40 mA) in transmission geometry. A 0.5° slit, 4 mm mask and 0.4 radSoller slits with a focusing mirror were used on the incident beam. APIXcel^(3D) detector, placed on the diffracted beam, was fitted with areceiving slit and 0.04 rad Soller slits. The instrument is performancechecked using silicon powder on a weekly basis. The software used fordata collection was X'Pert Data Collector v. 5.3 and the data wereanalyzed and presented using Diffrac Plus EVA v. 15.0.0.0 or HighscorePlus v. 4.5. Samples were prepared and analyzed in either a metal orMillipore 96 well-plate in transmission mode. X-ray transparent film wasused between the metal sheets on the metal well-plate and powders(approximately 1-2 mg) were used as received. The Millipore plate wasused to isolate and analyze solids from suspensions by adding a smallamount of suspension directly to the plate before filtration under alight vacuum.

The scan mode for the metal plate used the gonio scan axis, whereas a 2θscan was utilized for the Millipore plate. A performance check wascarried out using silicon powder (metal well-plate). The details of thedata collection were an angular range of 2.5 to 32.0° 2θ, a step size of0.0130° 2θ, and a total collection time of 2.07 minutes.

Samples were also collected on a Bruker D8 diffractometer using Cu K□radiation (40 kV, 40 mA), θ-2θ goniometer, and divergence of V4 andreceiving slits, a Ge monochromator and a Lynxeye detector. Theinstrument is performance checked using a certified Corundum standard(NIST 1976). The software used for data collection was DiffracPlus XRDCommander v2.6.1 and the data were analyzed and presented using DiffracPlus EVA v15.0.0.0.

Samples were run under ambient conditions as flat plate specimens usingpowder as received. The sample was gently packed into a cavity cut intopolished, zero-background (510) silicon wafer. The sample was rotated inits own plane during analysis. The details of the data collection werean angular range of 2 to 42° 2θ, a step size of 0.05° 2θ, and collectiontime of 0.5 s/step.

Example 13. Synthesis of Amorphous Compound 2

A 250 mL flask was charged with MeOH (151 mL) and the solution wascooled to 0-5° C. A concentrated solution of H₂SO₄ was added dropwiseover 10 minutes. A separate flask was charged with Compound 1 (151 g)and acetone (910 mL), and the H₂SO₄/MeOH solution was added dropwise at25-30° C. over 2.5 hours. A large amount of solid was precipitated.After the solution was stirred for 12-15 hours at 25-30° C., the mixturewas filtered, washed with MeOH/acetone (25 mL/150 mL), and dried at55-60° C. in vacuum to afford Compound 2 (121 g, 74%).

Analytic Method for Compound 2: The purity of Compound 2 was obtainedusing an Agilent 1100 HPLC system with a Waters XTerra Phenyl 5 μm4.6*250 mm column with the following conditions: 1 mL/min flow rate,read at 254 nm, 30° C. column temperature, 10 μL injection volume, and a30 minute run time. The sample was dissolved in ACN:water (90:10, v/v).The Gradient method for separation is shown below. R_(t) (min) ofCompound 2 was approximately 12.0 minutes.

Time (min) 0.1% H₃PO₄ in Water (A) % Acetonitrile (B) % 0 90 10 20 20 8020.1 90 10 30 90 10¹HNMR: (400 MHz, DMSO-d₆): δ 8.41 (br, 1H), 7.97 (s, 1H), 7.36 (t, J=8.0Hz, 2H), 7.22 (d, J=8.0 Hz, 2H), 7.17 (t, J=8.0 Hz, 1H), 6.73 (s, 2H),6.07 (d, J=8.0 Hz, 1H), 6.00 (dd, J=12.0, 8.0 Hz, 1H), 5.81(br, 1H),4.84-4.73 (m, 1H), 4.44-4.28 (m, 3H), 4.10 (t, J=8.0 Hz, 2H), 3.85-3.74(m, 1H), 2.95 (s, 3H), 1.21 (s, J=4.0 Hz, 3H), 1.15-1.10 (m, 9H).

Example 14. Characterization of Compound 2

Compound 2 was further characterized by eye, ¹HNMR, ¹³CNMR, ¹⁹FNMR, MS,HPLC, and XRPD (FIG. 15B). Residual solvent was measured by GC. Watercontent was measured by Karl Fischer Titration, and the water contentwas only 0.70%. Data is summarized in Table 18.

TABLE 18 Summary of Additional Characterization Data of Compound 2 TestResult Appearance White Solid NMR ¹HNMR peaks are listed in Example 4 MSMS(ESI+ve) [M + H]⁺ = 582.3 - conforms to structure HPLC 99.8% by AUC at254 nm (average of two preparations) Residual Methanol - 57 ppm Solventby GC Acetone - 752 ppm Dichloromethane - 50 ppm Ethyl Acetate - 176 ppmWater Content 0.70%

Example 15. Solubility of Compound 1 and Compound 2

Compound 1 and Compound 2 were both tested for solubility in biorelevanttest medias, including simulated gastric fluid (SGF), fasted-statesimulated gastric fluid (FaSSIF), and fed-state gastric fluid (FeSSIF).Results for Compound 1 are shown in Table 19 and results for Compound 2are shown in Table 20. Samples were stirred at room temperature (20-25°C.). Compound 2 was more than 40-fold more soluble than Compound 1 inwater at 2 hours and more than 25-fold more soluble at 24 hours. In SGFconditions, Compound 2 had a solubility of 84.2 mg/mL at 24 hourscompared to the solubility of 15.6 mg/mL of Compound 1 at the same timepoint. Compound 2 was also more soluble at 2 hours in the SGF conditionsthan Compound 1, and soluble enough to allow for testing even after 48hours while testing at 48 hours was not done with Compound 1.

TABLE 19 Compound 1 solubility testing results Solubility (in mg/mL)Descriptive Test Media 2 hours 24 hours Appearance term Water 1.5 2.5Clear Solution* Slightly Soluble SGF 13.8 15.6 Clear Solution Sparinglywith gum at the Soluble bottom FaSSIF 1.7 1.7 Turbid Slightly SolubleFeSSIF 2.8 2.9 Turbid Slightly Soluble *Sample appeared to be clear, yeta solubility of only 1.5 mg/mL was achieved. Upon further investigation,it was noted that a gummy film formed on the stir bar. The compound 1active pharmaceutical ingredient formed a gummy ball in diluent (90%water/10% acetonitrile) during standard preparation which required along sonication time to dissolve completely.

TABLE 20 Compound 2 solubility testing results Test Solubility (in mg/mLsalt base) Descriptive Media 2 hours 24 hours 48 hours Appearance termWater 65.3 68.0 N/A Turbid Soluble SGF 89.0 84.2 81.3 Turbid SolubleDescriptive FaSSIF 1.9 2.0 N/A Turbid Slightly Soluble FeSSIF 3.3 3.4N/A Turbid Slightly Soluble

Example 16. Chemical Stability of Compound 2

Compound 2 was tested for chemical stability at 25 and 40° C. over a 6month time period by monitoring organic purity, water content, ¹HNMR,DSC, and Ramen IR. The container closure system for the study was acombination medicinal valve bag with a pharmaceutical laminated filmover the pouch and desiccant silica gel between the two layers. Compound2 (1 g) was measured into each container. Bags were then stored at 25°C./60% RH (relative humidity) and 40° C./75% RH (relative humidity).Organic purity, water content, ¹HNMR, DSC and Raman were measured atTime 0, Month 1, Month 2, Month 3 and Month 6.

The purity of Compound 2 was obtained using a Shimadzu LC-20AD systemwith a Waters XTerra Phenyl, 5 μm, 4.6×250 mm column with the followingconditions: 1 mL/min flow rate, read at 254 nm, 35° C. columntemperature, and 10 μL injection volume. The sample was dissolved inacetonitrile—water (90:10) (v/v). The gradient method is shown below.

Time (min) A % (ACN) B % (water) 0 90 10 20 20 80 20.1 90 10 30 90 10

The water content of Compound 2 (250 mg) was determined by a watertitration apparatus using the Karl Fischer titration method.

Results are shown in Table 21 and Table 22. When Compound 2 was storedfor 6 months at 25 and 40° C., the rate of degradation was minimal. At 3months, Compound 2 was 99.75% percent pure at the 25° C. conditions and99.58% pure at the 40° C. conditions. At 6 months, Compound 2 was still99.74% pure at the 25° C. conditions and 99.30% pure at the 40° C.conditions. At 25° C., the percent of degradation product increased from0.03% at Day 0 to 0.08% after 6 months. At 40° C., the percent ofdegradation product increased from 0.03% to 0.39%. Over the course of 6months, the percent of water increased approximately 0.6% at 25° C. andincreased approximately 0.7% at 40° C.

Characterization by ¹HNMR, Raman, and DSC of Compound 2 at 1, 2, 3, and6 months was the same as the characterization of Compound 2 on day 0 atboth temperature conditions (Table 22), highlighting the long-termstability of Compound 2.

TABLE 21 Compound 2 rate of degradation over 6 months at 25 and 40° C.Percent of Maximum Time Percent Percent Degradation Impurity TestedWater Purity Product Percent 25° C. Day 0 1.2 99.82 0.03 0.12 Month 11.9 99.77 0.04 0.12 Month 2 1.8 99.75 0.06 0.12 Month 3 1.8 99.75 0.060.12 Month 6 1.8 99.74 0.08 0.13 40° C. Day 0 1.2 99.82 0.03 0.12 Month1 2.0 99.71 0.09 0.12 Month 2 1.9 99.63 0.15 0.12 Month 3 1.9 99.58 0.200.12 Month 6 1.9 99.30 0.39 0.14

TABLE 22 Characterization of Compound 2 during degradation study TimeTested ¹HNMR Raman DSC 25° C. Day 0 Initial Test Initial Test InitialTest Month 1 The same as Day 0 The same as Day 0 The same as Day 0 Month2 The same as Day 0 The same as Day 0 The same as Day 0 Month 3 The sameas Day 0 The same as Day 0 The same as Day 0 Month 6 The same as Day 0The same as Day 0 The same as Day 0 40° C. Day 0 Initial Test InitialTest Initial Test Month 1 The same as Day 0 The same as Day 0 The sameas Day 0 Month 2 The same as Day 0 The same as Day 0 The same as Day 0Month 3 The same as Day 0 The same as Day 0 The same as Day 0 Month 6The same as Day 0 The same as Day 0 The same as Day 0

Additional chemical stability studies of Compound 2 were measured todetermine the impurity and water levels. Three conditions were tested:accelerated stability (40±2° C./75±5% RH) over a 6-month time period,ambient stability (25±2° C./60±5% RH) over a 9-month period, andstability under refrigerator conditions (5±3° C.) over a 9-month timeperiod. The results for accelerated stability, ambient stability, andrefrigerator conditions are shown in Table 23, Table 24, and Table 25,respectively. Based on the results of these studies, Compound 2 is verychemically stable.

In the accelerated stability study (Table 23), at each time point(1^(st) month, 3^(rd) month, and 6^(th) month) where Compound 2 wasmeasured, the appearance of Compound 2 was always a white solid and theIR matched the reference standard. After six months, the total relatedsubstance 1 impurities was only 0.08% and there was no detection ofrelated substance 2 and isomers.

TABLE 23 Accelerated Stability (40 ± 2° C./75 ± 5% RH) of Compound 2Testing time point Items Specification 0 month 1^(st) month 3^(rd) month6^(th) month Appearance White or off- White White White White whitesolid solid solid solid solid IR correspond correspond / correspondcorrespond with reference with reference with reference with referencestandard standard standard standard Water ≤2.0% 0.45% 0.21% 0.36% 0.41%Related Impurity A ≤0.15% N.D. N.D. N.D. N.D. Substance 1 Impurity B≤0.15% N.D. N.D. N.D. N.D. Impurity F ≤0.15% N.D. N.D. N.D. 0.01%Impurity H ≤0.15% N.D. N.D. N.D. N.D. Any other single ≤0.10% 0.01%0.02% 0.01% 0.05% impurity Total Impurities ≤0.2% 0.01% 0.02% 0.02%0.08% Related Impurity G ≤0.15% N.D. N.D. N.D. N.D. Substance 2 IsomerImpurity C ≤0.15% N.D. / N.D. N.D. Impurity D ≤0.15% N.D. / N.D. N.D.Impurity E ≤0.15% N.D. / N.D. N.D. Assay 98.0%~102.0% 98.8%  101.5%  99.6%  99.5%  Microbial TAMC ≤1000 cfu/g <1 cfu/g / / / Testing Mold andYeast  ≤100 cfu/g <1 cfu/g / / / E. Coli Not Detected N.D. / / / N.D.:Not Detected

In the ambient stability study where the appearance, IR, water andimpurity levels were measured for nine months, the appearance ofCompound 2 was always a white solid and the IR always corresponded withthe reference sample. The results (Table 24) highlight how chemicallystable Compound 2 is. After 9 months, the percentage of water in thesample was only 0.20% and the total related substance 1 impurities wasonly 0.02%. Similarly to the accelerated stability studies, relatedsubstance 2 and any isomers of Compound 2 were not detected.

TABLE 24 Ambient stability (25 ± 2° C./60 ± 5% RH) of Compound 2 Testingtime point Item Specification 0 month 1^(st) month 3^(rd) month 6^(th)month 9^(th) month Appearance White or off- White White White WhiteOff-white white solid solid solid solid solid solid IR correspondcorrespond / correspond correspond correspond with reference withreference with reference with reference with reference standard standardstandard standard standard Water ≤2.0% 0.45% 0.19% 0.29% 0.46% 0.20%Related Impurity A ≤0.15% N.D. N.D. N.D. N.D. N.D. Substance 1 ImpurityB ≤0.15% N.D. N.D. 0.03% N.D. N.D. Impurity F ≤0.15% N.D. N.D. 0.02%0.01% N.D. Impurity H ≤0.15% N.D. N.D. N.D. N.D. N.D. Any other single≤0.10% 0.01% 0.01% 0.03% 0.02% 0.02% impurity Total Impurities ≤0.2%0.01% 0.02% 0.11% 0.05% 0.02% Related Impurity G ≤0.15% N.D. N.D. N.D.N.D. N.D. Substance 2 Isomer Impurity C ≤0.15% N.D. / N.D. N.D. N.D.Impurity D ≤0.15% N.D. / N.D. N.D. N.D. Impurity E ≤0.15% N.D. / N.D.N.D. N.D. Assay 98.0%~102.0% 98.8%  101.1%   99.6%  99.7%  100.9%  Microbial TAMC ≤1000 cfu/g <1 cfu/g / / / / Testing Mold and Yeast  ≤100cfu/g <1 cfu/g / / / / E. Coli Not Detected N.D. / / / / N.D.: NotDetected

The results of measuring the stability under refrigerator conditions areshown in Table 25. The only impurities detected even after 9 months werethose from related substance 1 and water. The water content after 9months was 0.32% and the total impurities of related substance 1 wereonly 0.01% of the sample. Compound 2 is very chemically stable underrefrigerator conditions.

TABLE 25 Stability under refrigerator conditions (5 ± 3° C.) of Compound2 Testing time point Item Specification 0 month 1^(st) month 3^(rd)month 6^(th) month 9^(th) month Appearance White or off- White WhiteWhite White Off-white white solid solid solid solid solid solid IRcorrespond correspond / correspond correspond correspond with referencewith reference with reference with reference with reference standardstandard standard standard standard Water ≤2.0% 0.45% 0.19% 0.32% 0.42%0.32% Related Impurity A ≤0.15% N.D. N.D. N.D. N.D. N.D. Substance 1Impurity B ≤0.15% N.D. N.D. 0.01% N.D. N.D. Impurity F ≤0.15% N.D. N.D.N.D. N.D. N.D. Impurity H ≤0.15% N.D. N.D. N.D. N.D. N.D. Any othersingle ≤0.10% 0.01% 0.01% 0.01% 0.01% 0.01% impurity Total Impurities≤0.2% 0.01% 0.01% 0.03% 0.03% 0.01% Related Impurity G ≤0.15% N.D. N.D.N.D. N.D. N.D. Substance 2 Isomer Impurity C ≤0.15% N.D. / N.D. N.D.N.D. Impurity D ≤0.15% N.D. / N.D. N.D. N.D. Impurity E ≤0.15% N.D. /N.D. N.D. N.D. Assay 98.0%~102.0% 98.8%  101.1%   100.2%   98.6% 101.4%   Microbial TAMC ≤1000 cfu/g <1 cfu/g / / / / Testing Mold andYeast  ≤100 cfu/g <1 cfu/g / / / / E. Coli Not Detected N.D. / / / /N.D.: Not Detected

Example 17. Plasma Levels of Metabolites Following Single Oral Doses ofCompound 2

A single oral dose of Compound 2 was administered to rats, dogs, andmonkeys, and the plasma levels of certain metabolites shown in Scheme 1were measured.

The conversion of Compound 2 to Compound 1 and metabolite 1-7 are shownin Table 26 and the results for metabolite 1-8 and metabolite 1-2 areshown in Table 27. In rats, low levels of Compound 1 exposure wereobserved, but high levels of metabolite 1-7, the nucleoside metaboliteof the active triphosphate (metabolite 1-6), were observed. In monkeys,roughly dose-proportional exposures of Compound 1 were measured. Indogs, supra-proportional Compound 1 exposures, indicative of first-passmetabolic clearance in the liver, were measured. Throughout the study,significantly more vomiting in dogs (5/5 in high dose group) than inmonkeys (1/5 in high dose group) was observed.

TABLE 26 Plasma levels of Compound 1 and metabolite 1-7 after singleoral doses of Compound 2 Compound 1 Metabolite 1-7 Dose* C_(max) T_(max)AUC_(0-last) C_(max) AUC_(0-last) Species (mg/kg) (ng/mL) (hr)(hr*ng/mL) (ng/mL) (hr*ng/mL) Rat^(a) 500 70.5    0.25 60.9 748 12000Dog^(b) 30 1530 0.25-1  1300 783 9270 100 8120 0.5-1 10200 2030 24200300 21300 204 44300 4260 60800 Monkey^(b) 30 63.5 0.5-2 176 42.5 1620100 783  1-2 1100 131 3030 300 501 204 1600 93.6 3660 3 males per doseper species; *dose formulations: ^(a)0.5% CMC, 0.5% Tween 80 in water;^(b)powder in capsules

TABLE 27 Plasma levels of metabolites 1-8 and 1-2 after single oral doseof Compound 2 Metabolite 1-8 Metabolite 1-2 Dose* C_(max) AUC_(0-last)C_(max) AUC_(0-last) Species (mg/kg) (ng/mL) (hr * ng/mL) (ng/mL) (hr *ng/mL) Rat^(a) 500 5060 35100 9650 20300 Dog^(b) 30 291 905 196 610 1001230 4370 886 2830 300 5380 35300 2380 8710 Monkey^(b) 30 209 5690 3001730 100 406 12300 1350 8160 300 518 16800 1420 11400 3 males per doseper species; *dose formulations: ^(a)0.5% CMC, 0.5% Tween 80 in water;^(b)powder in capsules

Example 18. Tissue Exposure of Active Triphosphate Following Compound 2Oral Dose

Heart and liver tissue levels of the active triphosphate (TP) ofCompound 2 (metabolite 1-6) were measured 4 hours after oral doses ofCompound 2. Samples of liver and heart were obtained at 4 hours after asingle dose of Compound 2, flash-frozen, homogenized and analyzed byLC-MS/MS for intracellular levels of the active TP. Tissue levels weremeasured in rats, dogs, and monkeys as shown in FIG. 16A. High levels ofthe active TP were measured in the liver of all species tested.Relatively low levels of the active TP were measured in the hearts ofdogs due to saturation of first-pass hepatic metabolism, andunquantifiable levels of TP were measured in rat and monkey hearts,indicative of liver-specific formation of the active TP. While notshown, compared to Compound 1 dosing, Compound 2 dosing improved TPdistribution.

Example 19. Pharmacological Comparison of Compound 1 and Compound 2 inDogs

A head-to-head comparison of dogs dosed with Compound 1 and Compound 2was conducted. The study measured plasma levels of Compound 1 andmetabolite 1-7 (from Scheme 1) out to 4 hours after dosing with Compound1 (25 mg/kg) and Compound 2 (30 mg/kg) (Table 28), and the AUC_((0-4hr))of metabolite 1-7 was twice as great with Compound 2 compared toCompound 1. Dose-normalized exposures to Compound 1 and metabolite 1-7are shown in Table 28. Values for AUC_((0-4hr)) for Compound 1,metabolite 1-7, and the sum of Compound 1+metabolite 1-7 were greaterafter dosing with Compound 2.

TABLE 28 Comparison of Plasma Levels following dosing with Compound 1and Compound 2 Mean Dose-normalized AUC_((0-4hr)) ^(a) (μM * hr) for:Compound 1 + Dosed Compound Compound 1 Metabolite 1-7 Metabolite 1-7Compound 1 (25 mg/kg) 0.2 1.9 2.1 Compound 2 (30 mg/kg) 1.0 4.1 5.1^(a)AUC_((0-4hr)) values normalized to a dose of 25 mg/kg

Liver/heart ratio triphosphate concentrations indicate that dosing withCompound 2, as compared to Compound 1, increases the selective deliveryof the triphosphate to the liver, as shown in Table 29. TheAUC_((0-4hr)) of the active guanine metabolite (1-6) afteradministration of Compound 1 measured in the heart was 174 μM*hr, whilethe AUC_((0-4hr)) of the active guanine metabolite (1-6) afteradministration of Compound 2 measured in the heart was 28 μM*hr. Theliver/heart ratio for Compound 2 was 20 compared to a liver/heart ratioof 3.1 for Compound 1.

TABLE 29 Comparison of Liver and Heart Exposure following dosing withCompound 1 and Compound 2 Mean Dose-normalized AUC_((0-4hr)) ^(a) (μM *hr) for: Dosed Compound Liver Heart Liver/Heart Compound 2 565   28^(b)20 Compound 1 537 174 3.1 ^(a)Active TP concentrations (1-6; Scheme 1)normalized to a dose of 25 mg/kg ^(b)Extrapolated below the lower limitof quantitation of the calibration curve

The effect of increased selectivity for the liver over the heart whenCompound 2 was administered compared to Compound 1 is also shown in FIG.16B. The heart and liver tissue levels of the active triphosphatefollowing a dosage of Compound 2 (30 mg/kg) were compared to the tissuelevels of the active triphosphate following a dosage of Compound 1 (25mg/kg). The concentration of the active TP was higher in the liver thanthe heart for both Compound 1 and Compound 2, but the active TP was moreselective for the liver over the heart when Compound 2 was dosedcompared to Compound 1.

Example 20. Plasma Profiles of Compound 2 Metabolites in Rats andMonkeys

Male Sprague-Dawley rats and cynomolgus monkeys (3 animals per dosegroup) were given single oral doses of Compound 2. Aliquots of plasmaprepared from blood samples treated with Dichlorvos were analyzed byLC-MS/MS for concentrations of Compound 1 and metabolite 1-7 (thenucleoside metabolite of the active triphosphate of Compound 2 shown inScheme 1), and pharmacokinetic parameters were determined usingWinNonlin. The results for a single 500 mg/kg dose in rats is shown inFIG. 17 and the results for a single 30, 100, or 300 mg/kg dose inmonkeys is shown in FIG. 18. The results are also summarized in Table30.

High plasma levels of metabolite 1-7, the nucleoside metabolite of theactive triphosphate (TP) of Compound 2, are indicative of formation ofhigh levels of the TP, even in rats where very low plasma levels ofparent nucleotide prodrug are observed due to the short half-life ofCompound 1 in rat blood (<2 min). Persistent plasma levels of metabolite1-7 reflect the long half-life of the TP.

In monkeys, plasma exposures (AUC) of Compound 1 were roughlydose-proportional, while metabolite 1-7 exposures were somewhat lessthan dose-proportional, although AUC values for both parent drug and thenucleoside metabolite of the active TP continue to increase up to thehighest dose tested (300 mg/kg).

Oral administration of Compound 2 in rats and monkeys produced high anddose-dependent plasma exposures to metabolite 1-7 (the nucleosidemetabolite of the intracellular active triphosphate of Compound 2);metabolite 1-7 exposures continued to increase up to the highest dosetested, reflecting substantial formation of the active TP in thesespecies.

TABLE 30 Plasma levels of Compounds 1 and 1-7 after single oral dose ofCompound 2 Species Rat^(a) Monkey^(b) Dose (mg/kg) 500 30 100 300Compound 1 C_(max) (ng/mL) 60.8 63.5 783 501 T_(max) (hr) 0.25 0.5-2  1-2 204 AUC_(0-last) 78.2 176 1100 1600 (hr * ng/mL) Metabolite 1-7C_(max) (ng/mL) 541 42.5 131 93.6 AUC_(0-last) 9640 1620 3030 3660 (hr *ng/mL) T_(max) (hr) 6-8 12-24 4 4-24 T_(1/2) (hr) 15.3 11.5 15.0 18.8dose formulations: ^(a)0.5% CMC, 0.5% Tween 80 in water; ^(b)powder incapsules

Example 21. The Effect of the Active Triphosphate of Compound 1 andCompound 2 on Mitochondrial Integrity

The relative efficiency of incorporation of the active triphosphate (TP)of Compound 1 and Compound 2, metabolite 1-6 (Scheme 1), by humanmitochondrial RNA polymerase was compared to the relative efficiency ofthe active TP of sofosbuvir and the active TP of INX-189. Compound 1 andCompound 2 are not likely to affect mitochondrial integrity since theiractive triphosphate is poorly incorporated by human mitochondrial RNApolymerase with an efficiency similar to that of the triphosphate ofsofosbuvir; the relative efficiency of incorporation of the triphosphateof INX-189 was up to 55-fold greater. Results are shown in Table 31. Theincorporation of these analogs by human mitochondrial RNA-dependent RNApolymerase (POLRMT) were determined according to Arnold et al.(Sensitivity of Mitochondrial Transcription and Resistance of RNAPolymerase II Dependent Nuclear Transcription to AntiviralRibonucleotides. PLoS Pathog., 2012, 8, e1003030).

TABLE 31 Kinetic Parameters for Nucleotide Analogs Evaluated with HumanMitochondrial RNA Polymerase K_(pol/Kd,app) Relative Nucleotide AnalogK_(pol) (s⁻¹) K_(d,app) (μM) (μM⁻¹s⁻¹) Efficiency* 2′-deoxy-2′-F-2′-0.00034 ± 0.00005  590 ± 250 5.8 × 10⁻⁷ ± 1.0 × 10⁻⁶ C-methyl UTP 2.6 ×10⁻⁷ (active TP of sofosbuvir) 2′-C-methyl GTP 0.051 ± 0.002 240 ± 262.1 × 10⁻⁴ ± 5.5 × 10⁻⁵ (active TP of INX- 0.2 × 10⁻⁴ 189) Active TP of0.0017 ± 0.0002 204 ± 94 8.3 × 10⁻⁶ ± 2.2 × 10⁻⁶ Compound 1 and 4.0 ×10⁻⁶ Compound 2 (metabolite 1-6) *Relative efficiency =(K_(pol)/K_(d,app))_(analog nucleotide)/(K_(pol)/K_(d,app))_(natural nucleotide)

Example 22. Activity of Compound 1 against Replicons Containing the NS5BSequence

A panel of replicons containing the NS5B sequences from various HCVgenotypes derived from 6 laboratory reference strains (GT1a, 1b, 2a, 3a,4a and 5a) (FIG. 19) and from 8 HCV patient plasma samples (GT1a, 1b,2a, 2b, 3a-1, 3a-2, 4a and 4d) (FIG. 20) were used to determine thepotency of Compound 1 and sofosbuvir.

Compound 1 was more potent than sofosbuvir against clinical andlaboratory strains of HCV. Compound 1 showed potent pan-genotypicantiviral activity in vitro against wild-type clinical isolates withEC₉₅ <80 nM, which is 4- to 14-fold more potent than sofosbuvir. Asshown in FIG. 20, EC₉₅ values for Compound 1 were 7-33 times lower thansofosbuvir against clinical isolates of all HCV genotypes tested. EC₅₀values for Compound 1 were 6-11 times lower than sofosbuvir againstlaboratory strains of HCV Genotypes 1-5 (FIG. 19).

Example 23. Single Ascending Dose (SAD) Study of Compound 2 in HealthyVolunteers (Part A) and GT1-HCV Infected Patients (Part B)

Compound 2 was tested in a single ascending dose (SAD) study to measureits safety, tolerability, and pharmacokinetic in healthy subjects (PartA). Part A was a randomized, double-blind, placebo-controlled SAD study.Healthy subjects in Part A received a single dose of Compound 2 orplacebo in the fasting state. Subjects were confined to the clinic fromDay −1 to Day 6.

Dosing in each cohort was staggered such that 2 subjects (1 active:1placebo) were evaluated for 48 hours after dosing before the remainderof the cohort was dosed. Each cohort received Compound 2 in ascendingorder. Dosing of sequential cohorts occurred based on review ofavailable safety data (through Day 5) and plasma pharmacokinetic data(through 24 h) of the prior cohort.

Dose escalation proceeded following satisfactory review of these data.As pharmacokinetic and safety data emerged from prior cohorts, dosesevaluated in Cohorts 3a-4a were adjusted by increments no more than 100mg. The total maximum dose evaluated in Part A did not exceed 800 mg.The dosing regimen for Part A is shown in Table 32.

TABLE 32 Dosing Regimen for Compound 2 Administration Part A of StudyCohort Population N (active:placebo) Compound 2 (Compound 1)* 1a Healthy6:2  50 (45) mg × 1 day 2a Healthy 6:2 100 (90) mg × 1 day 3a Healthy6:2 200 (180) mg × 1 day 4a Healthy 6:2 400 (360) mg × 1 day *Clinicaldoses are expressed in terms of Compound 2, with the approximateCompound 1 base equivalent in parenthesis

Healthy volunteers in the Part A portion of the study were male andfemale subjects between the ages of 18 and 65. Active and placeborecipients were pooled within each Part A cohort to preserve the studyblind.

Compound 2 was also tested in a single ascending dose (SAD) study tomeasure its safety, tolerability, pharmacokinetic, and antiviralactivity in GT1-HCV infected patients (Part B). Subjects in Part Breceived a single dose of Compound 2 in the fasting state. Patients wereconfined to the clinic from Day −1 to Day 6.

Part B was initiated after the safety (through Day 5) and plasmapharmacokinetic (through 24 h) data review from Cohort 3a in Part A.Available safety data (through Day 5) and pharmacokinetic data (through24 h) was reviewed for the first cohort in Part B (Cohort 1b) beforeenrolling subsequent Part B cohorts. Subsequent Part B cohorts were onlydosed following review of available safety and pharmacokinetic data fromthe respective doses in Part A as well as available safety (through Day5) from the prior Part B cohorts.

Dose escalation up to 600 mg in HCV-infected patients proceededfollowing satisfactory review of these data. The dosing regimen for PartB is shown in Table 33.

TABLE 33 Dosing Regimen for Compound 2 in Part B of Study CohortPopulation N (active) Compound 2 (Compound 1)* 1b GT1 HCV-Infected 3 100(90) mg × 1 day 2b GT1 HCV-Infected 3 300 (270) mg × 1 day 3b GT1HCV-Infected 3 400 (360) mg × 1 day 4b GT1 HCV-Infected 3 600 (540) mg ×1 day *Clinical doses are expressed in terms of Compound 2, with theapproximate Compound 1 base equivalent in parenthesis.

Patients infected with HCV were treatment-naïve, non-cirrhoticGT1-infected subjects with a viral load of ≥5 log₁₀ IU/mL.

No serious adverse events were recorded and no prematurediscontinuations were required in either Part A or Part B. All adverseeffects were mild to moderate in intensity and no dose-related patterns,including laboratory parameters, vital signs, and ECGs were evident.

Example 24. Results of the Single Ascending Dose (SAD) Study of Compound2

Pharmacokinetic of Compound 1 and nucleoside metabolite 1-7 weremeasured following the single dose of Compound 2. The C₂₄ trough plasmaconcentrations (C_(24h)) of metabolite 1-7 in HCV-infected patientsfollowing a 600 mg dose of Compound 2 was 25.8 ng/mL, which is more thandouble the plasma concentration dose following a 300 mg dose of Compound2. Metabolite 1-7 (shown in Scheme 1) can only be generated viadephosphorylation of the intracellular phosphate metabolite 1-4,metabolite 1-5, and metabolite 1-6, which is the active species.Therefore, metabolite 1-7 can be considered a surrogate of the activespecies. The pharmacokinetic data for all cohorts is shown in Table 34and Table 35. Values are reported as mean ±SD, except tor T_(max) wheremedian (range) is reported. Pharmacokinetic parameters were comparablein healthy and HCV-infected patients.

TABLE 34 Human Pharmacokinetic of Compound 1 and Metabolite 1-7 afterAdministration of a single dose of Compound 2 in Healthy Volunteers DoseC_(max) T_(max) AUC_(tot) T_(1/2) C_(24 h) (mg) (ng/mL) (h) (ng*h/mL)(h) (ng/mL) Part A, Healthy Subjects Compd 1 50 46.4 ± 17.6 0.5(0.5-0.5) 36.4 ± 12.3 0.32 ± 0.02 — 100  156 ± 96.3 0.5 (0.5-1.0) 167 ±110 0.53 ± 0.24 — 200 818 ± 443 0.5 (0.5-3.0) 656 ± 255 0.71 ± 0.16 —400 1194 ± 401  0.5 (0.5-1.0) 1108 ± 326  0.86 ± 0.15 — Metabolite 5027.9 ± 5.62 3.5 (3.0-4.0)  285 ± 69.4 7.07 ± 4.59 2.28 ± 0.95 1-7 10056.6 ± 14.0 4.0 (3.0-6.0) 663 ± 242 17.7 ± 14.7 4.45 ± 1.87 200  111 ±38.8 5.0 (3.0-6.0) 1524 ± 497  15.9 ± 7.95 13.7 ± 5.09 400  153 ± 49.46.0 (4.0-8.0) 2342 ± 598  15.6 ± 6.37 23.5 ± 6.31 *Based on 24-hrprofile.

TABLE 35 Human Pharmacokinetic of Compound 1 and Metabolite 1-7 afterAdministration of Compound 2 in GT1-HCV Infected Patients Dose C_(max)T_(max) AUC_(tot) T_(1/2) C_(24 h) (mg) (ng/mL) (h) (ng*h/mL) (h)(ng/mL) Compd 1 100  212 ± 32.0 0.5 (0.5-1.0)  179 ± 54.4 0.54 ± 0.12 —300 871 ± 590 0.5 (0.5-1.0) 818 ± 475 0.64 ± 0.20 — 300 2277 ± 893  0.5(0.5-1.0) 1856 ± 1025 0.84 ± 0.18 — 400 2675 ± 2114 1.0 (1.0-2.0) 2408 ±1013 0.86 ± 0.18 — 600 3543 ± 1649 1.0 (0.5-1.0) 4132 ± 1127 0.70 ± 0.13— Metabolite 100 50.2 ± 15.4 6.0 (4.0-6.0)  538 ± 103*  8.4 ± 4.3* 3.60± 0.40 1-7 300 96.9 ± 38.9 6.0 (3.0-6.0) 1131 ± 273*  8.1 ± 2.4* 10.9 ±3.51 300  123 ± 16.6 4.0 (3.0-6.0) 1420 ± 221  — 18.0 ± 8.83 400  160 ±36.7 4.0 (4.0-4.0) 2132 ± 120  11.6 ± 1.21 22.5 ± 3.29 600  198 ± 19.34.0 (4.0-6.0) 2176 ± 116  — 25.8 ± 4.08 *Based on 24-hr profile.

The mean plasma concentration-time profiles of Compound 1 and metabolite1-7 were also calculated for all cohorts of Part A and Part B of thestudy. FIG. 21 is the mean plasma-concentration of Compound 1 followinga single dose of Compound 2 and FIG. 22 is the mean plasma-concentrationof metabolite 1-7 following a single dose of Compound 2. As shown inFIG. 21, Compound 1 was quickly absorbed and rapidly/extensivelymetabolized in all cohorts from Part B. As shown in FIG. 22, metabolite1-7 was a major metabolite and exhibited sustained plasmaconcentrations. Plasma exposure of Compound 1 was dose-related whileexposure of metabolite 1-7 was dose-proportional.

For the HCV-infected subjects of Part B, measurements of HCV RNAquantitation were performed before, during, and after administration ofCompound 2. Plasma HCV RNA determinations were performed through the useof a validated commercial assay. Baseline was defined as the mean of Day−1 and Day 1 (pre-dose). A single 300 mg dose of Compound 2 (equivalentto 270 mg of Compound 1) resulted in significant antiviral activity inGT1b-HCV infected subjects. The mean maximum HCV RNA reduction 24 hourspost-dose following a single 300 mg dose was 1.7 log₁₀ IU/mL and thiscompares to a −2 log₁₀ IU/mL reduction after 1 day of 400 mg ofsofosbuvir monotherapy in GT1a HCV-infected subjects. The mean maximumHCV RNA reduction 24 hours post-dose following a single 100 mg dose was0.8 log₁₀ IU/mL. The mean maximum HCV RNA reduction was 2.2 log₁₀ IU/mLfollowing a single 400 mg dose. Individualpharmacokinetic/pharmacodynamic analyses for the individual subjectsfrom Part B of the study are shown in FIGS. 23A-23F. Metabolite 1-7concentration is plotted against HCV RNA reduction concentration, and asshown in FIGS. 23A-23F, plasma HCV RNA reduction correlates with plasmametabolite 1-7 exposure. Viral response is sustained with metabolite 1-7plasma concentrations that are greater than the EC₉₅ value against GT1b.The correlation between plasma concentration and HCV RNA reductionlevels indicates that a more profound response will be achievable withhigher doses of Compound 2.

Example 25. Predicted Steady-State Trough Levels of Metabolite 1-7exceed Compound 1 EC₉₅ Values against Clinical Isolates of HCV GT 1-4

As shown in FIG. 24, the steady-state trough plasma levels (C_(24,ss))of metabolite 1-7 following Compound 2 dosing in humans (600 mg QD (550mg free base equivalent) and 450 mg QD (400 mg free base equivalent))was predicted and compared to the EC₉₅ of Compound 1 in vitro across alltested clinical isolates to determine if the steady state plasmaconcentration is consistently higher than the EC₉₅, which would resultin high efficacy against any or all tested clinical isolates in vivo.The EC₉₅ for Compound 1 is the same as the EC₉₅ of Compound 2. ForCompound 2 to be effective, the steady-state trough plasma level ofmetabolite 1-7 should exceed the EC₉₅.

As shown in FIG. 24, the EC₉₅ of Compound 2 against all tested clinicalisolates ranged from approximately 18 to 24 nM.

As shown in FIG. 24, Compound 2 at a dose of 450 mg QD (400 mg free baseequivalent) in humans of provides a predicted steady state trough plasmaconcentration (C_(24,ss)) of approximately 40 ng/mL. Compound 2 at adose of 600 mg QD (550 mg free base equivalent) in humans of provides apredicted steady state trough plasma concentration (C_(24,ss)) ofapproximately 50 ng/mL.

Therefore, the predicted steady state plasma concentration of surrogatemetabolite 1-7 is almost double the EC₉₅ against all tested clinicalisolates (even the hard to treat GT3a), which indicates superiorperformance.

In contrast, the EC₉₅ of the standard of care nucleotide sofosbuvirranges from 50 to 265 nM across all tested HCV clinical isolates, withan EC₉₅ less than the predicted steady state concentration at thecommercial dosage of 400 mg for only two isolates, GT2a and GT2b. TheEC₉₅ for the commercial dosage of 400 mg of sofosbuvir is greater thanthe predicted steady state concentration for other clinical isolates,GT1a, GT1b, GT3a, GT4a, and GT4d.

The Compound 2 450 mg steady state trough plasma concentration(C_(24,ss)) was predicted using the 300 mg steady state trough plasmaconcentration (C_(24,ss)). The mean steady state trough plasmaconcentration (C_(24,ss)) at 300 mg was 26.4 ng/mL, and therefore thecalculation was 26.4*450/300=39.6 ng/mL.

The 600 mg steady state trough plasma concentration (C_(24,ss)) waspredicted using three approaches: 1) the 600 mg Day 1 C₂₄ mean was 25.8ng/mL and a 60% increase was assumed for reaching steady state.Therefore the calculation was 25.8*1.6=41.3 ng/mL; 2) the 400 mg day 1C₂₄ mean was 22.5 ng/mL and a 60% increase was assumed for reachingsteady state. Taking dose proportional PK into account, the calculationwas 22.5*1.6*600/400=54 ng/mL; and 3) the 300 mg steady state troughplasma concentration (C_(24,ss)) was 26.4 ng/mL and a proportional PKwas assumed. Therefore the calculation was 26.4*2=52.8 ng/mL. The 600 mgsteady state trough plasma concentration (C_(24,ss)) is the average ofthe 3 data points ((41.3+54+52.8)/3=49.3 ng/mL). There is generallyabout a 60% increase in C₂₄ at steady state compared to C₂₄ following asingle dose.

The data comparing the efficacy and pharmacokinetic steady stateparameters in FIG. 24 clearly demonstrates the unexpected therapeuticimportance of Compound 2 for the treatment of hepatitis C. In fact, thepredicted steady-state plasma level after administration of Compound 2is predicted to be at least 2-fold higher than the EC₉₅ for allgenotypes tested, and is 3- to 5-fold more potent against GT2. This dataindicates that Compound 2 has potent pan-genotypic antiviral activity inhumans. As shown in FIG. 24, the EC₉₅ of sofosbuvir at GT1, GT3, and GT4is greater than 100 ng/mL. Thus surprisingly, Compound 2 is activeagainst HCV at a dosage form that delivers a lower steady-state troughconcentration (40-50 ng/mL) than the steady-state tough concentration(approximately 100 ng/mL) achieved by a similar dosage form ofsofosbuvir.

Example 26. Formulation Description and Manufacturing of Compound 2

A representative non-limiting batch formula for Compound 2 tablets (50mg and 100 mg) is presented in Table 36. The tablets were produced froma common blend using a direct compression process as shown in FIG. 25.The active pharmaceutical ingredient (API) is adjusted based on theas-is assay, with the adjustment made in the percentage ofmicrocrystalline cellulose. The API and excipients (microcrystallinecellulose, lactose monohydrate, and croscarmellose sodium) werescreened, placed into a V-blender (PK Blendmaster, 0.5L bowl) and mixedfor 5 minutes at 25 rpm. Magnesium Stearate was then screened, added andthe blend was mixed for an additional 2 minutes. The common blend wasdivided for use in producing 50 mg and 100 mg tablets. The lubricatedblend was then compressed at a speed of 10 tablets/minutes using asingle punch research tablet press (Korsch XP1) and a gravity powderfeeder. The 50 tablets were produced using round standard concave 6 mmtooling and 3.5 kN forces. The 100 mg tablets were produced using 8 mmround standard concave tooling and 3.9-4.2 kN forces.

TABLE 36 Formulation of 50 mg and 100 mg Compound 2 Tablets % Mg/unitRaw Material w/w g/batch 50 mg Tablet 100 mg Tablet Compound 2 50.0180.0 50.0 100.0 Microcrystalline 20.0 72.0 20.0 40.0 Cellulose, USP/NF,EP Lactose Monohydrate, 24.0 86.4 24.0 48.0 USP/NF, BP, EP, JPCroscarmellose Sodium, 5.0 18.0 5.0 10.0 USP/NF, EP Magnesium Stearate,1.0 3.6 1.0 2.0 USP/NF, BP, EP JP Total 100.0 200.0

Compound 2 was adjusted based on the as-is assay, with the adjustmentmade in the percentage of microcrystalline cellulose. Compound 2 andexcipients (microcrystalline cellulose, lactose monohydrate, andcroscarmellose sodium) were screened, placed into a V-blender (PKBlendmaster, 0.5L bowl) and mixed for 5 minutes at 25 rpm. Magnesiumstearate was then screened, added and the blend was mixed for anadditional 2 minutes. The common blend was divided for use in producing50 mg and 100 mg tablets. The lubricated blend was then compressed at aspeed of 10 tablets/minutes using a single punch research tablet press(Korsch XP1) and a gravity powder feeder. The 50 mg tablets wereproduced using round standard concave 6 mm tooling and 3.5 kN forces.The 100 mg tablets were produced using 8 mm round standard concavetooling and 3.9-4.2 kN forces. The specifications of the 50 mg and 100mg tablets are shown in Table 37.

TABLE 37 Specifications of 50 mg and 100 mg Tablets of Compound 2 50 mgTablets 100 mg Tablets Average Weight (n = 10)  100 ± 5 mg 200 ± 10 mgIndividual Weight 100 ± 10 mg 200 ± 20 mg Hardness 5.3 kp 8.3 kpDisintegration <15 minutes <15 minutes Friability NMT 0.5% NMT 0.5%

The 50 mg and 100 mg tablets produced as described above were subjectedto 6 month stability studies under three conditions: 5° C.(refrigeration), 25° C./60% RH (ambient), and 40° C./75% RH(accelerated). Both the 50 mg and 100 mg tablets were chemically stableunder all three conditions tested.

Under refrigeration conditions (5° C.), both the 50 mg and 100 mgtablets remained white solids that did not change in appearance from T=0to T=6 months. Throughout the 6-month study, no impurities were reportedthat were greater than 0.05% for either the 50 mg tablets or the 100 mgtablets. The water content after 6 months was also less than 3.0% w/wfor both tablets. Similar results were reported when the tablets weresubjected to ambient conditions (25° C./60% RH); no impurities that weregreater than 0.05% were reported throughout the 6 months for bothtablets and the water content did not exceed 3.0% w/w at the 6-monthmark. When the tablets were subjected to accelerated conditions (40°C./75% RH), the appearance of the 50 mg and 100 mg tablets did notchange from a white, round tablet. One impurity was reported after 3months, but the impurity was only 0.09%. A second impurity was reportedafter 6 months, but the total impurity percentage was only 0.21% forboth the 50 mg and 100 mg tablets. Water content was 3.4% w/w at 6months for the 50 mg tablets and 3.2% w/w for the 100 mg tablets.

In a separate study, the stability of 50 mg and 100 mg tablets ofCompound 2 at ambient conditions (25° C./60% RH) was measured over 9months. The appearance of the 50 mg and 100 mg tablet did not changefrom a white round tablet over the course of 9 months. Impurities in the50 mg tablet were less than 0.10% after 9 months and impurities in the100 mg tablet were less than 0.05%. The water content of the 50 mgtablet and the 100 mg tablet after 9 months was only 2.7% w/w and 2.6%w/w, respectively.

This specification has been described with reference to embodiments ofthe invention. However, one of ordinary skill in the art appreciatesthat various modifications and changes can be made without departingfrom the scope of the invention as set forth in the claims below.Accordingly, the specification is to be regarded in an illustrativerather than a restrictive sense, and all such modifications are intendedto be included within the scope of invention.

What is claimed is:
 1. A hemi-sulfate salt form of a compound of theformula:


2. The hemi-sulfate salt form of the compound of claim 1, of theformula:


3. A solid dosage form comprising the hemi-sulfate salt form of theformula:


4. A solid dosage form comprising the hemi-sulfate salt form of theformula:


5. The solid dosage form of claim 4 that provides a steady-state troughplasma level (C_(24,ss)) of metabolite

between approximately 20-60 ng/mL.
 6. The solid dosage form of claim 5,wherein the steady-state trough plasma level (C_(24,ss)) is betweenapproximately 20-50 ng/mL.
 7. The solid dosage form of claim 5, whereinthe steady-state trough plasma level (C_(24,ss)) is betweenapproximately 20-30 ng/mL.
 8. The solid dosage form of claim 5, whereinthe steady-state trough plasma level (C_(24,ss)) is betweenapproximately 20-25 ng/mL.
 9. The solid dosage form of claim 5, whereinthe area under the curve of metabolite

is between approximately 1,500 ng*h/mL and 3,000 ng*h/mL.
 10. The soliddosage form of claim 9, wherein the area under the curve is betweenapproximately 1,800 ng*h/mL and 3,000 ng*h/mL.
 11. The solid dosage formof claim 9, wherein the area under the curve is between approximately2,400 ng*h/mL and 3,000 ng*h/mL.
 12. The solid dosage form of claim 9,wherein the area under the curve is between approximately 2,700 ng*h/mLand 3,000 ng*h/mL.
 13. The solid dosage form of claim 9, wherein thearea under the curve is between approximately 2,000 ng*h/mL and 2,200ng*h/mL.
 14. A pharmaceutical composition comprising the hemi-sulfatesalt form of a compound of the formula


15. A pharmaceutical composition comprising the hemi-sulfate salt formof a compound of the formula


16. The pharmaceutical composition of claim 14, in a solid dosage formthat delivers at least 300 mg, at least 400 mg, or at least 500 mg ofthe compound.
 17. The pharmaceutical composition of claim 14, in a soliddosage form that delivers at least 600 mg of the compound.
 18. Thepharmaceutical composition of claim 14, in a solid dosage form thatdelivers at least 700 mg of the compound.
 19. The pharmaceuticalcomposition of claim 15, in a solid dosage form that delivers at least300 mg, at least 400 mg, or at least 500 mg of the compound.
 20. Thepharmaceutical composition of claim 15, in a solid dosage form thatdelivers at least 600 mg of the compound.
 21. The pharmaceuticalcomposition of claim 15, in a solid dosage form that delivers at least700 mg of the compound.
 22. The pharmaceutical composition of claim 14,wherein the pharmaceutically acceptable carrier is suitable for oraldelivery.
 23. The pharmaceutical composition of claim 15, wherein thepharmaceutically acceptable carrier is suitable for oral delivery. 24.The pharmaceutical composition of claim 22, wherein the pharmaceuticallyacceptable carrier is in the form of a tablet or a capsule.
 25. Thepharmaceutical composition of claim 23, wherein the pharmaceuticallyacceptable carrier is in the form of a tablet or a capsule.
 26. A methodto treat a hepatitis C infection in a human in need thereof, comprisingadministering an effective amount of a compound of the formula


27. A method to treat a hepatitis C infection in a human in needthereof, comprising administering an effective amount of a compound ofthe formula


28. The method of claim 26, wherein the compound is administered orally.29. The method of claim 27, wherein the compound is administered orally.30. The method of claim 26, wherein at least 300 mg, at least 400 mg, orat least 500 mg of the compound is administered.
 31. The method of claim26, wherein at least 600 mg of the compound is administered.
 32. Themethod of claim 26, wherein at least 700 mg of the compound isadministered.
 33. The method of claim 27, wherein at least 300 mg, atleast 400 mg, or at least 500 mg of the compound is administered. 34.The method of claim 27, wherein at least 600 mg of the compound isadministered.
 35. The method of claim 27, wherein at least 700 mg of thecompound is administered.
 36. The method of claim 26, wherein thecompound is administered for at least 6 weeks.
 37. The method of claim26, wherein the compound is administered for at least 8 weeks.
 38. Themethod of claim 26, wherein the compound is administered for at least 12weeks.
 39. The method of claim 27, wherein the compound is administeredfor at least 6 weeks.
 40. The method of claim 27, wherein the compoundis administered for at least 8 weeks.
 41. The method of claim 27,wherein the compound is administered for at least 12 weeks.
 42. Themethod of claim 26, wherein the compound is administered once a day. 43.The method of claim 26, wherein the compound is administered every otherday.
 44. The method of claim 27, wherein the compound is administeredonce a day.
 45. The method of claim 27, wherein the compound isadministered every other day.